Time In Primary Culture Research Articles

The impact of the sample time of secondary bacterial culture on the risk of exposure to contaminated platelet components: A mathematical analysis.

The US Food and Drug Administration (FDA) issued a guidance for bacterial risk control strategies for platelet components in September 2019 that includes strategies using secondary bacterial culture (SBC). While an SBC likely increases safety, the optimal timing of the SBC is unknown. Our aim was to develop a model to provide insight into the best time for SBC sampling. We developed a mathematical model based on the conditional probability of a bacterial contamination event. The model evaluates the impact of secondary culture sampling time over a range of bacterial contamination scenarios (lag and doubling times), with the primary outcome being the optimal secondary sampling time and the associated risk. Residual risk of exposure decreased with increasing inoculum size, later sampling times for primary culture, and using higher thresholds of exposure (in colony-forming units per milliliter). Given a level of exposure, the optimal sampling time for secondary culture depended on the timing of primary culture and on the expiration time. In general, the optimal sampling time for secondary culture was approximately halfway between the time of primary culture and the expiration time. Our model supports that the FDA guidance is quite reasonable and that sampling earlier in the specified secondary culture windows may be most optimal for safety.

Glycolysis-Optimized Conditions Enhance Maintenance of Regenerative Integrity in Mouse Spermatogonial Stem Cells during Long-Term Culture.

SummaryThe application of spermatogonial stem cell (SSC) transplantation for regenerating male fertility requires amplification of SSC number in vitro during which the integrity to re-establish spermatogenesis must be preserved. Conventional conditions supporting proliferation of SSCs from mouse pups have been the basis for developing methodology with adult human cells but are unrefined. We found that the integrity to regenerate spermatogenesis after transplantation declines with advancing time in primary cultures of pup SSCs and that the efficacy of deriving cultures from adult SSCs is limited with conventional conditions. To address these deficiencies, we optimized the culture environment to favor glycolysis as the primary bioenergetics process. In these conditions, regenerative integrity of pup and adult SSCs was significantly improved and the efficiency of establishing primary cultures was 100%. Collectively, these findings suggest that SSCs are primed for conditions favoring glycolytic activity, and matching culture environments to their bioenergetics is critical for maintaining functional integrity.

CD11d β2 integrin expression on human NK, B, and γδ T cells.

The CD11d integrin is expressed on the cell surface of leukocytes that belong to the myeloid lineage, but its expression on lymphocytes remains unexplored. To test the hypothesis that CD11d is expressed on lymphocyte subsets, we employed a multicolor flow cytometry panel to identify CD11d expression on B, NK, CD4+ and CD8+ αβ T cells (αβTc), and γδ T cells (γδTc) in human PBMC samples. CD11d was highly expressed on NK cells, B cells, and γδTc, but not αβTc. CD11d expression was higher on freshly isolated γδTc compared with αβTc from healthy donors, yet both inter- and intradonor variability was evident. Over time in primary culture, we consistently observed higher CD11d levels on γδTc compared with αβTc from the same donor. Furthermore, CD11d expression on γδTc increased over time and correlated with levels of IL-2 supplementation. Of interest, a greater percentage of Vδ1 γδTc expressed CD11d than did Vδ2 γδTc, which suggested differential roles for this integrin that may segregate with γδTc subsets. These results expand the potential for CD11d to regulate lymphocyte migration and tissue retention, and illuminate the possibility of a previously unconsidered role for CD11d in leukocyte biology and disease.

Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties

BackgroundThe treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed.ResultsAt first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion.ConclusionsBoth isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.

Optimization of culture of mesenchymal stem cells: a comparison of conventional plate and microcarrier cultures

There has been increasing interest in mesenchymal stem cells (MSCs) because of their potential use for regenerative therapy; however, there is no well-defined protocol for MSCs culture. This study compares techniques of conventional plate and microcarrier culturing of MSCs. Here, different conditions for isolation and expansion of rat MSCs have been examined and it was found that plating density and plating time in primary culture played important roles for culture of these rat MSCs. When plated at 10(8) /cm(2) density for 72 h, in primary culture, recycling stem cells (RS cells) predominated, and characteristics of rat MSCs (including morphology, growth rate, phenotype and differentiation potentials) remained stable during expansion until passage 14. For subculture of the cells, it was found that their growth rate when incubated at 33 °C was higher than those incubated at 37 °C, and maximal increase was 10- and 6-fold respectively. When cultured using microcarriers, at a density of 1 × 10(5) /mg beads, growth kinetics, phenotype and differentiation potentials also remained constant for cells between passage 2nd and 14th; their maximal number increased 16-fold. Compared to conventional plate culture, culture using gelatine porous microcarrier Cultispher-S was superior for large-scale production of rat MSCs.

Optimisation of culture and cardiogenic differentiation of rats mesenchymal stem cells

There has been an increasing interest in mesenchymal stem cells (MSCs) because of their potential use for regenerative therapy, especially for cardiovascular disease, however there is still no well-defined protocol...

Establishment and cryopreservation of liver, heart and muscle cell lines derived from the Chinese alligator (Alligator sinensis)

The Chinese alligator, Alligator sinensis, is a critically endangered species. A conservation project of gene resources for an endangered species first involves the preservation of organs, tissues, gametes, genomic DNA libraries and cell lines. The present study is the first to establish and cryopreserve cell lines of liver, heart and muscle tissues from the Chinese alligator. The study revealed that there was a large discrepancy in cell migration time in primary cultures among liver (11–12 d), heart (13–14 d) and muscle (17–18 d) tissue pieces. The differences in time in primary cell culture suggested that it was relatively easy to build visceral-derived cell lines for reptiles. Biological analysis showed that the population doubling time for thawed cells was approximately 36 h. Karyotyping revealed that the frequency of Chinese alligator cells showing chromosome number as 2n=32 was 88.6%–93.4%. Chinese alligator cell lines established here provide a vital resource for research and are likely to be useful for protection of this rare and critically endangered species. Furthermore, the establishment of these methods may supply technical and theoretical support for preserving genetic resources at the cellular level for other reptile species.

Stability of the contractile assembly and Ca2+-activated tension in adenovirus infected adult cardiac myocytes.

Adenovirus-mediated gene transfer into adult cardiac myocytes in primary culture is a potentially useful method to study the structure and function of the contractile apparatus. However, the consequences of adenovirus infection on the highly differentiated state of the cultured myocyte have not been determined. We report here a detailed analysis of myofilament structure and function over time in primary culture and after adenovirus infection. Adult rat ventricular myocytes in primary culture were infected with a recombinant adenovirus vector expressing either the LacZ or alkaline phosphatase reporter gene. Control and infected myocytes were collected at days 0-7 post-isolation/infection, and myofilament isoform expression was determined by SDS-PAGE and Western blot. Laser scanning densitometry showed that the alpha- to beta-myosin heavy chain ratio, the stoichiometry of the myosin light chains and the expression of the adult troponin T isoform did not change over time in culture or with adenovirus treatment. Importantly, examination of Ca2+-activated tension in single myocytes showed no change in the shape or position of the tension-pCa relationship in the control and adenovirus infected myocytes during primary culture. These results indicate that the structure and function of adult cardiac myocytes are stable in short term primary culture and are not affected by adenovirus infection per se, and therefore provide the foundation for the use of adenovirus-mediated myofilament gene transfer to study contractile apparatus structure and function in adult cardiac myocytes.

Immunolocalization of Na+, K+-ATPase in the gill epithelium of rainbow trout, Oncorhynchus mykiss.

The Na+,K+-ATPase (the sodium pump) plays a crucial role in ion transport in the fish gill. An immunocytochemical method has been optimized, using the mouse monoclonal antibody IgG alpha5, raised against the alpha subunit of the avian sodium pump, to localize Na+,K+-ATPase in fish gill cells. The method appears to be successful for the immunolocalization of Na+,K+-ATPase in both paraffin-embedded gill tissue sections and primary cultures of fish gill epithelial cells. The immunostaining has demonstrated that Na+,K+-ATPase-positive cells are mainly localized on the primary lamellae, in the interlamellar region, which is in agreement with the distribution of ion-transporting cells, also called chloride cells, as shown by electron microscopy. Na+,K+-ATPase-positive cells have been demonstrated for the first time in primary cultures of gill epithelial cells. Comparative labeling studies of primary cultures have shown that sites of Na+,K+-ATPase-positive cells correspond to sites of cells labeled with dimethylaminostyrylmethyl-pyridiniumiodine, a fluorescent mitochondrial probe for ion-transporting cells. The immunocytochemical detection method for Na+,K+-ATPase in cells is proposed as an easy and specific Na+-transport-related method to characterize and localize ion-transporting cells in primary cultures and in tissue sections of fish gill epithelium.

Synthesis of fibronectin and laminin by type II pulmonary epithelial cells.

Previous investigations demonstrated that type II pulmonary epithelial cells regulate extracellular matrix deposition as a function of time in primary culture. In those studies, the matrix fraction was analyzed as a whole. The present work focused on two components of the type II cell matrix, fibronectin and laminin. These glycoproteins have differing effects on differentiation of type II cells in primary culture. Fibronectin synthesis was quantitated between day 1 and day 6 in the cells, matrix, and medium; laminin synthesis was quantitated only in the cells. Although total fibronectin synthesis was regulated as a function of time in culture, reaching its greatest value on day 2, the average proportion of newly synthesized fibronectin in the cells (35%), medium (50%), and matrix (15%) remained constant over a 6-day interval. Between day 2 and day 6, the relative abundance of fibronectin messenger RNA increased 6.5-fold. Rates of cellular laminin synthesis did not vary with time in culture. These results support a role for differential regulation of fibronectin and laminin synthesis to determine the composition of the type II cell extracellular matrix.

Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.

Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (> 97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a "spindle cell," consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic‐responsiveness in normal mouse mammary epithelial cells in vitro

Mammary epithelial cells were isolated from mid-pregnant BALB/c mice, grown within collagen gels and maintained on DME/F12 (1:1) media containing 10% bovine calf serum and 10 micrograms/ml insulin. Initial time-course and dose-response studies showed that epidermal growth factor (EGF)-induced autophosphorylation of the EGF-receptor (EGF-R) in these cells was maximal 5 min after exposure to 75 ng/ml EGF. Mammary epithelial cells displaying little or no growth during their first 2 days in primary culture cells were found to contain low levels of EGF-R. However, EGF-induced autophosphorylation of the EGF-R in these cells was extremely intense. Subsequent studies demonstrated that during the proliferative and plateau phases of growth, EGF-R levels progressively increased, while conversely EGF-induced autophosphorylation of the EGF-R decreased over time in primary culture. These results demonstrate that EGF-R levels and autophosphorylation do not show a direct correlation with mammary epithelial cell mitogen-responsiveness. Intense EGF-R autophosphorylation appears to be required for initiating growth, but sustained mammary epithelial cell proliferation occurs when EGF-R autophosphorylation is low. This inverse relationship between EGF-R levels and autophosphorylation may reflect changes in receptor affinity and function during the various phases of mammary epithelial cell growth in primary culture.

Transforming growth factor-beta (TGF-beta 1) inhibits pancreatic acinar cell growth.

Effects of transforming growth factor (TGF)-beta 1 on mouse pancreatic acinar cell growth and rapid intracellular responses to cholecystokinin (CCK) were examined in vitro. TGF-beta 1 inhibited [3H]thymidine incorporation stimulated by either the CCK analogue caerulein, epidermal growth factor, or insulin. TGF-beta 1 inhibition of growth stimulated by a maximal dose of caerulein (1 nM) was dose dependent with one-half maximal effects occurring at approximately 5 pM and maximal inhibition seen with 30 pM. In contrast to its effects on CCK-stimulated [3H]thymidine incorporation, TGF-beta 1 had no effect on CCK-stimulated increases in amylase release or intracellular Ca2+ concentration. To determine whether TGF-beta 1 might be an autocrine growth regulator, pancreatic mRNA was probed for the presence of TGF-beta 1 transcripts. TGF-beta 1 mRNA was not detected in whole pancreas but was detectable with increasing abundance over time in primary cultures of pancreatic acinar cells. The appearance of the TGF-beta 1 mRNA corresponded to the period of rapid cellular proliferation in vitro. These results suggest that TGF-beta 1 may be an autocrine growth inhibitor in the pancreas and that the inhibitory effects of TGF-beta 1 on pancreatic acinar cell growth occur at sites distal to those involved in stimulus-secretion coupling.

Responses of subcultured rat aortic smooth muscle myocytes to vasoactive agents and KCl-induced depolarization

We have developed a culture system in which a single-mass primary culture can be used for as long as 6 wk as a source of subcultured smooth muscle myocytes for the study of the changes of their shape upon addition of vasoactive agents (angiotensin, vasopressin, norepinephrine, and serotonin) and KCl depolarization. Responses of subcultivated myocytes were shown to be reproducible with time in primary culture before subculture and consistent with responses of thoracic aorta to the same agents. Effect of KCl depolarization could be blocked with calcium antagonist PN 200-110. Consistently, the presence of calcium L-channels was shown using whole cell patch-clamp recordings. A comparative study of the responses of myocytes derived from two different segments of the thoracic aorta showed that these cells displayed responses with different maximal amplitudes and the same potencies according to their topological origin in the vessel.

Phenotypic Changes of Rabbit Mandibular Condylar Cartilage Cells in Culture

The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 micrograms/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.

Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes.

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.

Real-time observations of microtubule dynamic instability in living cells.

Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.

Binding of α 2-macroglobulin to hepatocytes: Mechanism of in vivo clearance

The binding of 125I-labeled human α 2-macroglobulin-methylamine to adult rat hepatocytes in primary culture was studied at 4°C. Cells which had been in culture for 4 hours exhibited steady state ligand binding after 1 hour, a receptor number of 22,400 receptors per cell, and a dissociation constant of 0.6 nM. Adult rat hepatocytes exhibited a significant decrease in receptor number with increased time in primary culture with less than 10% of the initial number of receptors remaining after 2 days (p <0.01). In autopsy studies of mice injected intravenously with 125I-labeled α 2-macroglobulin-methylamine, greater than 90% of the cleared ligand was found in the liver. Autoradiography of the liver demonstrated that 80% of the ligand was cleared by hepatocytes. It is concluded that the hepatocytes are the primary pathway for clearance from the circulation of receptor recognized α 2-macroglobulin.

Differential response to hormones of defined distal nephron epithelia in culture.

Defined cultures of rabbit kidney cortical collecting tubule (CCT) and cortical thick ascending limb of Henle's loop (CAL) were grown in monolayers from individual microdissected tubules and maintained for up to five passages, a maximum of 53 days. CCT cells contained cytochemically demonstrable vasopressin-stimulated adenylate cyclase, whereas CAL cells were characterized by the localization of Na+-K+-ATPase. [3H]thymidine labeling index decreased with time in primary cultures in the presence or absence of 3% serum. When added to unsupplemented serum-free media alone or in combinations, the growth factors dexamethasone, thyroxine, insulin, epidermal growth factor, and prolactin stimulated [3H]thymidine incorporation to different extents. CCT cells were maximally stimulated by addition of dexamethasone alone, whereas a combination of dexamethasone, thyroxine, insulin, and prolactin was most stimulatory for CAL cells. Addition of hormones concerned with renal ion and water transport to fully supplemented serum-free media inhibited [3H]thymidine labeling index: 1) vasopressin, isoproterenol, and dibutyryl cAMP were equally inhibitory in CCT and CAL cultures; 2) parathyroid hormone and prostaglandin E1 were more inhibitory in CAL cultures; and 3) aldosterone was particularly inhibitory in CCT cultures.

Heterogeneity of Na +-independent 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and L-leucine transport in isolated rat hepatocytes in primary culture

Kinetic heterogeneity of saturable Na +-independent transport of both leucine and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid uptake by primary cultures of isolated rat hepatocytes was demonstrated. Such heterogeneity was not evident in the transformed hepatic cell line, HTC. One of the two components of Na +-independent transport in normal hepatocytes increased in activity with increasing time in primary culture, whereas the second component decreased in activity during the same time period. On the basis of kinetic and inhibition analysis the two components have tentatively been identified as Systems L and T . The increase in System T activity during the initial days of primary cultures of hepatocytes which is dependent on de novo synthesis of both RNA and protein, represents one of the first demonstrations of regulation for a Na +-independent amino acid transport system.