Long-term cell culture is an important biological approach but is also characterized by degeneration in cellular morphology, proliferation rate, and function. To explore this phenomenon in a systematic way, we conducted an integrative proteomics-metabolomics measurement of two cardiovascular cell lines of AC16 and HUVECs. The 18th culturing passages, i.e., G18, showed as the turning points by cell metabolism profiles, in which the metabolomic changes demonstrated the dysfunction of energy, amino acid, and ribonucleotide metabolism metabolic pathways. Although active protein networks showed mitochondria abundance AC16 and oxidative/nitrative sensitive HUVECs indicated the different degeneration patterns, the G18 and G30 proteomics evidenced the senescence by processes of signal transduction, signaling by interleukins, programmed cell death, cellular responses to stimuli, cell cycle, mRNA splicing, and translation. Some crucial proteins (RPS8, HNRNPR, SOD2, LMNB1, PSMA1, DECR1, GOT2, OGDH, PNP, CBS, ATIC, and IMPDH2) and metabolites (L-glutamic acid, guanine, citric acid, guanosine, guanosine diphosphate, glucose 6-phosphate, and adenosine) that contributed to the dysregulation of cellular homeostasis are identified by using the integrative proteomic-metabolomic analysis, which highlighted the increased cellular instability. These findings illuminate some vital molecular processes when culturing serial passages, which contribute holistic viewpoints of in vitro biology with emphasis on the replicative senescence of cardiovascular cells.

Volumetric muscle loss (VML) is a clinical state that results in impaired skeletal muscle function. Engineered skeletal muscle can serve as a treatment for VML. Currently, large biopsies are required to achieve the cells necessary for the fabrication of engineered muscle, leading to donor-site morbidity. Amplification of cell numbers using cell passaging may increase the usefulness of a single muscle biopsy for engineering muscle tissue. In this study, we evaluated the impact of passaging cells obtained from donor muscle tissue by analyzing characteristics of in vitro cellular growth and tissue-engineered skeletal muscle unit (SMU) structure and function. Human skeletal muscle cell isolates from three separate donors (P0-Control) were compared with cells passaged once (P1), twice (P2), or three times (P3) by monitoring SMU force production and determining muscle content and structure using immunohistochemistry. Data indicated that passaging decreased the number of satellite cells and increased the population doubling time. P1 SMUs had slightly greater contractile force and P2 SMUs showed statistically significant greater force production compared with P0 SMUs with no change in SMU muscle content. In conclusion, human skeletal muscle cells can be passaged twice without negatively impacting SMU muscle content or contractile function, providing the opportunity to potentially create larger SMUs from smaller biopsies, thereby producing clinically relevant sized grafts to aid in VML repair.

BACKGROUND: Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design.
RESULTS: The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates.
CONCLUSIONS: The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

This manuscript describes step-by-step procedures to establish and manage fresh and cryopreserved cultures of nerve-derived human Schwann cells (hSCs) at the desired scale. Adaptable protocols are provided to propagate hSC cultures through serial passaging and perform routine manipulations such as enzymatic dissociation, purification, cryogenic preservation, live-cell labeling, and gene delivery. Expanded hSCs cultures are metabolically active, proliferative, and phenotypically stable for at least three consecutive passages. Cell yields are expected to be variable as determined by the rate of growth of individual batches and the rounds of subculture. The purity, however, can be maintained high at >95% hSC regardless of passage. The cells obtained in this manner are suitable for various applications, including small drug screens, in vitro modeling of neurodevelopmental processes, and cell transplantation. One caveat of this protocol is that continued expansion of same-batch hSC populations is eventually restricted due to senescence-linked growth arrest.

Serial passaging is a fundamental technique in experimental evolution. The choice of bottleneck severity and frequency poses a dilemma: longer growth periods allow beneficial mutants to arise and grow over more generations, but simultaneously necessitate more severe bottlenecks with a higher risk of those same mutations being lost. Short growth periods require less severe bottlenecks, but come at the cost of less time between transfers for beneficial mutations to establish. The standard laboratory protocol of 24-h growth cycles with severe bottlenecking has logistical advantages for the experimenter but limited theoretical justification. Here we demonstrate that contrary to standard practice, the rate of adaptive evolution is maximized when bottlenecks are frequent and small, indeed infinitesimally so in the limit of continuous culture. This result derives from revising key assumptions underpinning previous theoretical work, notably changing the metric of optimization from adaptation per serial transfer to per experiment runtime. We also show that adding resource constraints and clonal interference to the model leaves the qualitative results unchanged. Implementing these findings will require liquid-handling robots to perform frequent bottlenecks, or chemostats for continuous culture. Further innovation in and adoption of these technologies has the potential to accelerate the rate of discovery in experimental evolution.

HIV-1 emerged from SIVcpz evolving in humans. Humanized mice are an effective tool for assessing viral evolution via measuring viral loads, CD4+ T cell decline, and analyzing genetic changes. Four serial passages showed many non-synonymous mutations important for the adaptation and evolution of SIVcpz to human immune cells.

HIV-2 Group F virus with an origin in NHPs was isolated from only two individuals. Two serial passages in hu-mice showed increased viral loads, CD4+ T cell decline and nonsynonymous genetic changes showing its capacity for further evolution, and spread in the human.

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes.

The passage number of cells refers to the number of subculturing processes that the cells have undergone. The effect of passage number on morphological and phenotypical characteristics of cells is of great importance. Advanced glycation end products have also been associated with cell functionality and characteristics. Murine monocyte RAW 264.7 cells differentiate into osteoclasts upon receptor activation caused by nuclear factor-kappa-Β ligand (RANKL) treatment. This study aims to identify the role of passage number on intracellular advanced glycation end products (AGEs) formation and osteoclastogenic differentiation of RAW 264.7 cells. Western blotting was performed to check intracellular AGE formation along with fluorometric analysis using a microplate reader. Tartrate-resistant acid phosphatase (TRAP) staining was performed to check osteoclastogenic differentiation, and qPCR was realized to check the responsible mRNA expression. Immunofluorescence was used to check the morphological changes. Intracellular AGE formation was increased with passaging, and the higher passage number inhibited multinucleated osteoclastogenic differentiation. Osteoclastogenic gene expression also showed a reducing trend in higher passages, along with a significant reduction in F-actin ring size and number. Lower passages should be used to avoid the effects of cell subculturing in in vitro osteoclastogenesis study using RAW 264.7 cells.