Monitoring and modeling density-dependent growth of anchorage-dependent cells

The density-dependent growth of Chinese hamster ovary (CHO) cells was monitored on-line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate cell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth.     

Human keratinocytes cultured without a feeder layer undergo progressive loss of differentiation markers.

Culture of keratinocytes in conventional medium without a mesenchyme-derived feeder layer leads to poor growth and impaired differentiation; however, the exact pathway and degree of differentiation achieved in such conditions is unclear. We have cultured normal human keratinocytes in Rheinwald and Green's medium, on plastic without a feeder layer, in order to investigate the degree of differentiation that they achieve in these conditions. Intermediate filament proteins, tonofibrils and desmosomes were assumed as markers of differentiation and their expression was analyzed by immunohistochemistry and electron microscopy. Before reaching confluence, keratinocytes expressed keratin molecules, as well as vimentin, and formed tonofibrils and desmosomes. The expression of these markers was progressively reduced until confluence and was totally lost thereafter, while cultures could be propagated for at least six passages. On the contrary, reseeding on a feeder layer after the first passage led to rapid cell death. It could be concluded that signals from a feeder layer are relevant to support continuous synthesis of intermediate filaments proteins and formation of tonofibils and desmosomes, and that the derangement of the cytoskeleton in these conditions leads to altered, not simply defective, response to delayed stimulation by a feeder layer.     

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.     

On-line determination of optimum time for switching from growth phase to production phase in tissue plasminogen activator production in a microcarrier cell culture

To maximize the productivity of tissue plasminogen activator (TPA) by a mammalian cell culture, on-line determination of the optimum time to switch from the cell growth phase to the TPA production phase was investigated. By measuring the TPA production activity of the cells during the cell growth culture, it was shown that this optimum time was not necessarily the same as the time at which the cell concentration was maximized, and that the optimum time varied with growth culture batch. The TPA production activity of the cells during the growth culture could be estimated by on-line regression analysis using physiological data of the current state, including the oxygen consumption rate (Io₂) and cell concentration, as well as data from past batches. Applying this on-line estimation, the optimum switching time was determined to be the time at which the TPA production activity of the cells in the growth culture became highest, or higher than a certain value according to determined criteria.     

Direct quantification of in vitro cell growth through image analysis

Summary Objective, accurate, non-intrusive measurement of in vitro cell growth was realized through microcomputerized video image analysis. Recently-released video and digitizing hardware and software were incorporated into an analytical system which accurately quantified visual differences between cultures on a cell number or fresh mass basis. Sequential measurements during culture incubation further detected and quantified subtle changes in colony area and density resulting from growth. Each measurement was acquired rapidly, without encroaching on the in vitro environment, so cell growth was undisturbed. Custom software routines coordinated the quantification of this detailed record into precise cumulative growth curves.     

Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium

A serum-free medium (LEP-1) has been developed for mouse epidermal keratinocytes. LEP-1 consists of "Ca2+-free" Eagle's MEM with non-essential amino acids and seven added supplements (transferrin, 5 micrograms/ml; epidermal growth factor (EGF), 5 ng/ml; hydrocortisone, 0.5 microM; insulin, 5 micrograms/ml; phosphoethanolamine and ethanolamine, each 50 microM; bovine pituitary extract, 180 micrograms of protein/ml). Although serum-free the culture system was dependent for growth on bovine pituitary extract as the only still undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was developed to investigate the action of growth factors, hormones and other supplements on keratinocytes. Cells grown in LEP-1 (calcium concentration was 0.03 mM) maintained a high proliferative rate and presented the typical morphology of basal epidermal cells. When the calcium concentration of the medium was raised to 1.0 mM, the cells were triggered to differentiate terminally. The epithelial nature of the cells was demonstrated both by electron microscopy and by immunostaining with anti-keratin antibody. The maturation stage of the keratinocytes was defined by several morphological features during the proliferative phase and in terminally differentiating cultures. This serum-free system supported a useful number of cell divisions while keratinocytes retained the capacity to undergo terminal differentiation when given the appropriate stimulus. It provides, therefore, provides a useful model for investigations on growth, differentiation and malignant transformation of epidermal cells in culture.     

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres ¹. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo ². The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.Download video file.^{(57M, mp4)}     

Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment

In the present study in vitro expansion of human keratinocytes by supplementing dermal fibroblasts conditioned medium (DFCM) has been reported. Effect of two different DFCM acquired by culturing fibroblasts in keratinocyte-specific medium (defined keratinocytes serum free medium, DFCM-DKSFM) and fibroblast-specific serum free medium (F12: DMEM nutrient mix, DFCM-FD) have been compared. Growth kinetics of keratinocytes in terms of efficiency of cell attachment, expansion index, apparent specific growth rate and growth potential at the end of culture was evaluated in culture supplemented with DFCM-DKSFM and DFCM-FD in comparison with control i.e. DKSFM only. Results indicated that supplementation of DFCM caused significant increase in keratinocyte attachment. Efficiency of keratinocyte attachment in culture supplemented with bFCM-DKSFM was significantly higher compared to those cultured in DFCM-FD and DKSFM. In addition, the expansion index of keratinocytes in cultures supplemented with DFCM-DKSFM and DFCM-FD were 3.7 and 2.2 times higher than that of control condition even though the apparent growth rate and proliferative potential was found significantly lower. These results suggested that supplementation of DFCM enhanced expansion of keratinocyte by increasing efficiency of cell attachment, and DFCM-DKSFM provided suitable condition for in vitro expansion of keratinocytes compared to DFCM-FD and control condition.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.     

杂交瘤细胞培养中摄氧速率(OUR)的在线检测及其与细胞生长及代谢的关系

在批式及灌流培养条件下研究了杂交瘤细胞在无血清培养基中的生长、代谢情况与氧消耗的关系。应用动力学方法在线进行OUR的检测,同时离线取样检测其他参数。结果发现OUR与谷氨酰胺的消耗、抗体的生成及活细胞密度间有明显的相关关系,进一步的分析还发现在对数生长期,OUR与活细胞密度间具有良好的线性关系,qOUR(0.103±0.028)×10^-12mol/cell/h,可以通过它来进行细胞密度的在线检测。并通过以ΔOUR=0时刻作为灌流调整点进行连续灌流培养的初步实验验证了OUR作为培养过程反馈控制参数的可能性。     

Development of an optical system for the non‐invasive tracking of stem cell growth on microcarriers

The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non‐invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi‐illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12‐day culture noted, prior to the onset of aggregation. The developed image acquisition system and post‐processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032–2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Impact of plasminogen on an in vitro wound healing model based on a perfusion cell culture system

Vascular reorganization in wound healing is a complex process, which involves coagulation, endothelial cell proliferation and migration, basement membrane regeneration, and fibrinolysis. During this healing process, the hemostatic system and the angiogenic system are intimately interconnected. To elucidate the contribution of plasminogen in the process of wound healing, we have established a perfusion cell culture system. Using this novel cell culture system, we found that addition of plasminogen in the perfusion medium allowed the "scratch-wounded" endothelial cells to recover completely, while mini-plasminogen only affected the migration but not the proliferation of the endothelial cells. In the process of recovery with the addition of plasminogen, significant plasmin activity could only be detected when the growth of the endothelial cells have almost reached confluence. This finding indicates that wound healing is triggered and promoted during the absence of the proteolytic activity of plasmin. In addition, we could not detect any matrix metalloproteinase activity in the perfusion culture medium throughout the whole culture period. However, we did found that the circulating medium collected from the perfusion system at the early phase of the healing process has stimulatory activity on the growth of endothelial cells, but the proliferative activity decreased back to the basal level when the cells reached confluence. Thus, by using the perfusion cell culture system, we found that proliferation of endothelial cells is regulated by plasminogen and the wound healing process is controlled by a temporal interaction between the endothelial cells and plasminogen.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures

Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor. Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells. Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms. Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file. At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies. The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording.     

AMPK regulation of the growth of cultured human keratinocytes

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). At concentrations of 10(-4) and 10(-3) M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10(-6) M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D3 (10(-7) and 10(-6) M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D3 is AMPK-independent.