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Abstract 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.

Developing mechanical energy harvesters for electrical stimulation (ES) needed to augment cell behavior is a burgeoning area of interest. Mechanical energy harvesters that can generate electrical energy in electrolyte‐containing aqueous environments offer a unique solution for delivering ES to cells. In this work, a fully integrated ES assembly (FESA) is introduced that comprises coiled polydopamine (PDA) containing carbon nanotube yarn (CNT) harvesters, serving as ES generators, and poly(3,4‐ethylenedioxythiophene) coated carbon nanotube (PEDOT/CNT) sheets employed as a conductive scaffold. The PDA containing CNT (PDA/CNT) yarn, a novel twistron electrode, achieves an enhanced electrical power at a lower matching impedance than coiled CNT yarn to efficiently transfer ES to the conductive scaffold. The PEDOT used for the scaffold provides a suitable surface for cell adhesion and low resistance for effective ES transmission. In addition, the upscaled array of coiled PDA/CNT yarns provides an ES current density range up to 75.4 µA cm−2, which is much higher than for ES systems using different mechanical energy harvesters. This FESA is designed to provide an optimal level of ES for the proliferation and differentiation of chondrocytes. The findings illuminate the potential of chemically modified twistron energy harvesters as an innovative and effective strategy to promote biological response. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Cells in the human body experience different growth environments and conditions, such as compressive pressure and oxygen concentrations, depending on the type and location of the tissue. Thus, a culture device that emulates the environment inside the body is required to study cells outside the body. Methods A blanket-type cell culture device (Direct Contact Pressing: DCP) was fabricated with an alginate-based hydrogel. Changes in cell morphology due to DCP pressure were observed using a phase contrast microscope. The changes in the oxygen permeability and pressure according to the hydrogel concentration of DCP were analyzed. To compare the effects of DCP with normal or artificial hypoxic cultures, cells were divided based on the culture technique: normal culture, DCP culture device, and artificial hypoxic environment. Changes in phenotype, genes, and glycosaminoglycan amounts according to each environment were evaluated. Based on this, the mechanism of each culture environment on the intrinsic properties of conserving chondrocytes was suggested. Results Chondrocytes live under pressure from the surrounding collagen tissue and experience a hypoxic environment because collagen inhibits oxygen permeability. By culturing the chondrocytes in a DCP environment, the capability of DCP to produce a low-oxygen and physical pressure environment was verified. When human primary chondrocytes, which require pressure and a low-oxygen environment during culture to maintain their innate properties, were cultured using the hydrogel blanket, the original shapes and properties of the chondrocytes were maintained. The intrinsic properties could be recovered even in aged cells that had lost their original cell properties. Conclusions A DCP culture method using a biomimetic hydrogel blanket provides cells with an adjustable physical pressure and a low-oxygen environment. Through this technique, we could maintain the original cellular phenotypes and intrinsic properties of human primary chondrocytes. The results of this study can be applied to other cells that require special pressure and oxygen concentration control to maintain their intrinsic properties. Additionally, this technique has the potential to be applied to the re-differentiation of cells that have lost their original properties.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition. [ABSTRACT FROM AUTHOR]

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration. The biological status of human adipose-derived mesenchymal stem cells (hADSCs) between the Petri dish culture system (PDCS) and the gel microsphere culture system (GMCS) was discovered in depth. hADSCs kept in an undifferentiation status with higher stemness in the GMCS than the PDCS through regulating cell-ECM interaction. Further, the effect of osteochondral regeneration of the bilayer scaffolds from the GMCS was better than that from the PDCS. [Display omitted] [ABSTRACT FROM AUTHOR]

Genetic recombination is an important evolutionary mechanism for RNA viruses and can facilitate escape from immune and drug pressure. Recombinant hepatitis C virus (HCV) variants have rarely been detected in patients, suggesting that HCV has intrinsic low recombination rate. Recombination of HCV has been demonstrated in vitro between non-functional genomes, but its frequency and relevance for viral evolution and life cycle has not been clarified. We developed a cell-based assay to detect and quantify recombination between fully viable HCV genomes, using the reconstitution of green fluorescent protein (GFP) as a surrogate marker for recombination. Here, two GFP-expressing HCV genomes carrying different inactivating GFP mutations can produce a virus carrying a functional GFP by recombining within the GFP region. Generated constructs allowed quantification of recombination rates between markers spaced 603 and 553 nucleotides apart by flow cytometry and next-generation sequencing (NGS). Viral constructs showed comparable spread kinetics and reached similar infectivity titers in Huh7.5 cells, allowing their use in co-transfections and co-infections. Single-cycle co-transfection experiments, performed in CD81-deficient S29 cells, showed GFP expression in double-infected cells, demonstrating genome mixing and occurrence of recombination. Quantification of recombinant genomes by NGS revealed an average rate of 6.1 per cent, corresponding to 49 per cent of maximum detectable recombination (MDR). Experiments examining recombination during the full replication cycle of HCV, performed in Huh7.5 cells, demonstrated average recombination rates of 5.0 per cent (40.0 per cent MDR) and 3.6 per cent (28.8 per cent MDR) for markers spaced by 603 and 553 nucleotides, respectively, supporting a linear relationship between marker distance and recombination rates. First passage infections using recombinant virus supernatant resulted in comparable recombination rates of 5.9 per cent (47.2 per cent MDR) and 3.5 per cent (28.0 per cent MDR), respectively, for markers spaced by 603 and 553 nucleotides. We developed a functional cell-based assay that, to the best of our knowledge, allows for the first time detailed quantification of recombination rates using fully viable HCV constructs. Our data indicate that HCV recombines at high frequency between highly similar genomes and that the frequency of recombination increases with the distance between marker sites. These results have implication for our understanding of HCV evolution and emphasize the importance of recombination in the reassortment of mutations in the HCV genome. [ABSTRACT FROM AUTHOR]

Two typical medium replacement methods are employed to culture cells, that is, traditional manual medium change, wherein complete medium changes are performed at short-term intervals; and perfusion, wherein continuous culture media delivery and discharge are performed. The former is associated with the issue of readily accumulation of metabolic waste products, which is resolved by the latter. However, the latter requires a specific chamber for cell culturing such as a microfluidic channel for flow stability. Generally, bio-medical researchers require a culturing process that utilizes commercial culture dishes to test their conventional manipulation know-how, experience, and protocols. Thus, we constructed a perfusion-culture system using commercial 35 mm culture dishes. To use commercial culture dishes, it is necessary to maintain the culturing conditions including a constant volume and uniform flow rate, and we invented a novel adapter, culture dish adapter (CD-Adapter), attached to a commercial culture dish. The CD-Adapter is fixed with a holding jig to keep the medium volume constant, so it can maintain a uniform flow in the culture dish. Additionally, we demonstrate the applicability of the system by applying it to a perfusion culture of 293T cells, revealing a 50% reduction in DDIT3, a cellular stress marker, compared with that in conventional manual medium change. [ABSTRACT FROM AUTHOR]

A ring pump (RP) is a useful tool for microchannels and automated cell culturing. We have been developing RPs (a full-press ring pump, FRP; and a mid-press ring pump, MRP). However, damage to cells which were sent by the RP and the MRP was not investigated, and no other studies have compared the damage to cells between RPs and peristaltic pumps (PPs). Therefore, first, we evaluated the damage to cells that were sent by a small size FRP (s-FRP) and small size MRPs (s-MRPs; gap = 25 or 50 μm, respectively). “Small size” means that the s-FRP and the s-MRPs are suitable for microchannel-scale applications. The survival rate of cells sent by the s-MRPs was higher than those sent by the s-FRP, and less damage caused by the former. Second, we compared the survival rate of cells that were sent by a large size FRP (l-FRP), a large size MRP (l-MRP) (gap = 50 μm) and a PP. “Large size” means that the l-FRP and the l-MRP are suitable for automated cell culture system applications. We could not confirm any differences among the cell survival rates. On the other hand, when cells suspended in Dulbecco’s phosphate-buffered saline solution were circulated with the l-MRP (gap = 50 μm) and the PP, we confirmed a difference in cell survival rate, and less damage caused by the former.

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5′UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5′UTR and NS3-3′UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5′UTR and 3′UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated “CH6acc”), releasing HCV of 104.3–104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >105 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 108 HEV genome RNA copies (103 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products. [ABSTRACT FROM AUTHOR]

Research on regenerative medicine and cell therapy has progressed in recent years. Numerous cultured cells are used directly in clinical settings in regenerative medicine and cell therapy. It is important to observe the proliferation of the cultured cells using a microscope. However, the removal of culture vessels from a CO2 incubator subjects the cells to stress due to environmental changes and adversely affects cell growth. Thus, we have developed a cell culture system equipped with automated observation function that allows automated observation of cell cultures without removing the cultured cells from the CO2 incubator. This system comprises an automated cell observation unit to observe the cultured cells and an automated transfer unit to transfer the culture vessels in the CO2 incubator since this environment is harsh for mechanical devices. The automated cell observation unit consists of some optical components that should be stored in a moisture-proof case to isolate them from the environment of the CO2 incubator. The automated transfer unit is verified to have sufficient durability using oil and grease that does not exhibit any effect on cell growth. This system can acquire some time-lapse images of the cell culture continuously in the CO2 incubator.

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Abstract: Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1–6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6–2.1log10 higher infectivity titers (4.7–6.2log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV. [Copyright &y& Elsevier]

Abstract: The purpose of this study is to demonstrate pH-responsive chitosan is able to control cell behavior in response to small changes in environmental pH, which is at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. HeLa cells attached and spread well on chitosan at pH 6.99 and 7.20. When the pH was increased to 7.65, over 90% of cells would rapidly detached from chitosan surface within 1 h. Similarly, fibronectin adsorbed on chitosan at pH 7.20 also rapidly desorbed after increasing the medium pH. Most importantly and interestingly, medium pH adjustment could be facilitated by altering environment pCO2. It was found over 80% of HeLa cells could be recovered from chitosan surface within 1 h and the viability of detached cells was more than 95% by transferring the culture plate from incubator to atmospheric condition. Additionally, chitosan substrate could effectively control attachment/detachment of various types of cells including cell lines HaCaT, H1299, NIH-3T3, and primary corneal fibroblasts, indicating the technology described here is easily reproducible and should be promising for controlling rapid fibronectin adsorption/desorption and cell attachment/detachment for tissue engineering applications. [Copyright &y& Elsevier]

Abstract: The 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) is structured into four domains (I–IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5′UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1–7 recombinants with highly diverse 5′UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5′UTR of genotypes 1–6 or with heterotypic chimeric (1a/3a and 3a/1a) 5′UTR domains I, II or III, and 1a recombinants with 5′UTR domain I of genotypes 1–6. All were fully functional in Huh7.5 cells; therefore, the 5′UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5′-terminus was required. We demonstrated that J6/JFH1 with 5′UTR of genotypes 1–6 responded similarly to interferon-α2b. This study provides novel insight into the role of the 5′UTR in the HCV life cycle and facilitates HCV basic research and testing of 5′UTR-targeting antivirals. [Copyright &y& Elsevier]

Chronic liver disease caused by infection with hepatitis C virus (HCV) is an important global health problem that currently affects 170 million people. A major impediment in HCV research and drug development has been the lack of culture systems supporting virus production. This obstacle was recently overcome by using JFH1-based full-length genomes that allow production of viruses infectious both in vitro and in vivo. Although this improvement was important, because of the restriction to the JFH1 isolate and a single chimera consisting of J6CF and JFH1-derived sequences, broadly based comparative studies between different HCV strains were not possible. Therefore, in this study we created a series of further chimeric genomes allowing production of infectious genotype (GT) 1a, 1b, 2a, and 3a particles. With the exception of the GT3a/JFH1 chimera, efficient virus production was obtained when the genome fragments were fused via a site located right after the first transmembrane domain of NS2. The most efficient construct is a GT2a/2a chimera consisting of J6CF- and JFH1-derived sequences connected via this junction. This hybrid, designated Jc1, yielded infectious titers 100- to 1,000-fold higher than the parental isolate and all other chimeras, suggesting that determinants within the structural proteins govern kinetic and efficiency of virus assembly and release. Finally, we describe an E1-specific antiserum capable of neutralizing infectivity of all HCV chimeras. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we constructed another HCV replicon from HC-J4, one of chimpanzee-infectious HCV clones. An HCV replicon derived from HC-J4 (RpJ4) consists of HCV-5′ untranslated region, neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural region, NS3 to NS5B, and HCV-3′ untranslated region. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDR mutant and RpJ4-S2201del/ISDR mutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). After transfection into Huh-7 cells and G418 selection, RpJ4 and RpJ4/ISDR mutants did not produce any colony. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDR mutant, with the RpJ4-S2201del/ISDR mutant being most efficient. Hence the HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhanced its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV replications. [ABSTRACT FROM AUTHOR]

A new cell culture system expressing the entire HCV genorne has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were transfected with a recombinant plasmid containing full-length HCV cDNA genome by using lipofectamine 2000, followed by infection with recombinant vaccinia virus vTF7-3 containing the T7 RNA polymerase gene. Synthesis of positive-strand HCV RNA could be detected in the transfected cells by RT-PCR. Western blot analysis revealed that HCV structural and non-structural proteins were correctly processed. In transfected HeLa cells 47 nm virus-like particles were assembled, which could be recognized by anti-HCV E2 antibodies. The titer of HCV was 107 copies/mL in our cell culture system, which was significantly higher than that of infected patients' sera and that from all reported cell culture systems. Supernatant from transfected HeLa cells were infectious to Huh7 cells and the titer of HCV was 106 copies/mL. Moreover, negative-strand RNA of HCV in Huh7 cells could be detected by using strand-specific RT-PCR, which demonstrated that replication of HCV occurred in the permissive cell lines. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that mechanical forces affect a wide range of cellular behaviors. These forces regulate important cellular responses in the human body and consist of gravity, hydrostatic pressure, stretch, and shear stress, which is exerted on the vascular system by the passage of blood flow. We reasoned that these forces might be significant and dynamic regulators of cellular functions within the human body. While cellular effects of stretch and shear stress have been studied particularly with endothelial cells, little is known about the effects of gravity and hydrostatic pressure to cells. To examine the direct effect of hydrostatic pressure, we developed a culture device to confer hydrostatic pressures to cells ranging from 0 to 1,000 psi. We subjected human neuroblastoma cells and rIL-2-activated lymphocytes to a constant pressure of 20 or 100 psi for 48 h and attempted to identify genes regulated by hydrostatic pressure. Genes of regulator of G-protein signaling 5 in neuroblastoma cells and CHC1-L in lymphocytes increased after exposure to hydrostatic pressure. The results demonstrated that hydrostatic pressure directly regulates the expression of specific genes in mammalian cells. Moreover, there may be some underlying mechanisms that have common effects in altered physical environments. Our in vitro culture system may provide some insight into the mechanisms through the intracellular processes affected by mechanical forces.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-γ (IFN-γ), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-γ secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-γ production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety. [ABSTRACT FROM AUTHOR]

Genetic recombination is an important evolutionary mechanism for RNA viruses and can facilitate escape from immune and drug pressure. Recombinant hepatitis C virus (HCV) variants have rarely been detected in patients, suggesting that HCV has intrinsic low recombination rate. Recombination of HCV has been demonstrated in vitro between non-functional genomes, but its frequency and relevance for viral evolution and life cycle has not been clarified. We developed a cell-based assay to detect and quantify recombination between fully viable HCV genomes, using the reconstitution of green fluorescent protein (GFP) as a surrogate marker for recombination. Here, two GFP-expressing HCV genomes carrying different inactivating GFP mutations can produce a virus carrying a functional GFP by recombining within the GFP region. Generated constructs allowed quantification of recombination rates between markers spaced 603 and 553 nucleotides apart by flow cytometry and next-generation sequencing (NGS). Viral constructs showed comparable spread kinetics and reached similar infectivity titers in Huh7.5 cells, allowing their use in co-transfections and co-infections. Single-cycle co-transfection experiments, performed in CD81-deficient S29 cells, showed GFP expression in double-infected cells, demonstrating genome mixing and occurrence of recombination. Quantification of recombinant genomes by NGS revealed an average rate of 6.1 per cent, corresponding to 49 per cent of maximum detectable recombination (MDR). Experiments examining recombination during the full replication cycle of HCV, performed in Huh7.5 cells, demonstrated average recombination rates of 5.0 per cent (40.0 per cent MDR) and 3.6 per cent (28.8 per cent MDR) for markers spaced by 603 and 553 nucleotides, respectively, supporting a linear relationship between marker distance and recombination rates. First passage infections using recombinant virus supernatant resulted in comparable recombination rates of 5.9 per cent (47.2 per cent MDR) and 3.5 per cent (28.0 per cent MDR), respectively, for markers spaced by 603 and 553 nucleotides. We developed a functional cell-based assay that, to the best of our knowledge, allows for the first time detailed quantification of recombination rates using fully viable HCV constructs. Our data indicate that HCV recombines at high frequency between highly similar genomes and that the frequency of recombination increases with the distance between marker sites. These results have implication for our understanding of HCV evolution and emphasize the importance of recombination in the reassortment of mutations in the HCV genome., (© The Author(s) 2022. Published by Oxford University Press.)

Fibroblast to myofibroblast differentiation is a key feature of wound-healing in soft tissues, including the vagina. Vaginal fibroblasts maintain the integrity of the vaginal wall tissues, essential to keep pelvic organs in place and avoid pelvic organ prolapse (POP). The micro-environment of vaginal tissues in POP patients is stiffer and has different extracellular matrix (ECM) composition than healthy vaginal tissues. In this study, we employed a series of matrices with known stiffnesses, as well as vaginal ECMs, in combination with vaginal fibroblasts from POP and healthy tissues to investigate how matrix stiffness and composition regulate myofibroblast differentiation in vaginal fibroblasts. Stiffness was positively correlated to production of α-smooth muscle actin (α-SMA). Vaginal ECMs induced myofibroblast differentiation as both α-SMA and collagen gene expressions were increased. This differentiation was more pronounced in cells seeded on POP-ECMs that were stiffer than those derived from healthy tissues and had higher collagen and elastin protein content. We showed that stiffness and ECM content regulate vaginal myofibroblast differentiation. We provide preliminary evidence that vaginal fibroblasts might recognize POP-ECMs as scar tissues that need to be remodeled. This is fundamentally important for tissue repair, and provides a rational basis for POP disease modelling and therapeutic innovations in vaginal reconstruction. [ABSTRACT FROM AUTHOR]

A ring pump (RP) is a useful tool for microchannels and automated cell culturing. We have been developing RPs (a full-press ring pump, FRP; and a mid-press ring pump, MRP). However, damage to cells which were sent by the RP and the MRP was not investigated, and no other studies have compared the damage to cells between RPs and peristaltic pumps (PPs). Therefore, first, we evaluated the damage to cells that were sent by a small size FRP (s-FRP) and small size MRPs (s-MRPs; gap = 25 or 50 μm, respectively). "Small size" means that the s-FRP and the s-MRPs are suitable for microchannel-scale applications. The survival rate of cells sent by the s-MRPs was higher than those sent by the s-FRP, and less damage caused by the former. Second, we compared the survival rate of cells that were sent by a large size FRP (l-FRP), a large size MRP (l-MRP) (gap = 50 μm) and a PP. "Large size" means that the l-FRP and the l-MRP are suitable for automated cell culture system applications. We could not confirm any differences among the cell survival rates. On the other hand, when cells suspended in Dulbecco's phosphate-buffered saline solution were circulated with the l-MRP (gap = 50 μm) and the PP, we confirmed a difference in cell survival rate, and less damage caused by the former. [ABSTRACT FROM AUTHOR]