A flow diffusion chamber for research on cells in culture

A flow diffusion chamber designed for studying cells and tissues in culture is described. The chamber contains a plate with a great number of isolated holes, which enables one to perform the cultivation of cells at different distances from the porous membrane separating the cells from the perfused medium. An individual porous membrane can be placed above each hole. Evidence for the selective permeability of domestic membranes under the conditions of cell culture in chamber is presented. The chamber makes possible a simultaneous cultivation of a great number of various cultures with different conditions of mass exchange with common perfused medium, which contributes to intensification of studies.     

The insulin secretion of a minced neonatal rat pancreas cultured in a pancreatic chamber, in response to various insulin secretagogues

The minced pancreas of the neonatal rat was cultured for 35 days in a pancreatic chamber which was constructed of a plastic tube and an ultrafiltration membrane. Insulin and amylase secreted from this pancreatic chamber into the culture medium were measured. During the experiment, the concentration of glucose in the culture medium was changed between 5.5 and 16.5 mM at 2-3 day intervals in order to determine the insulin secretory response of the pancreatic tissue. Insulin secretion was markedly increased in response to 16.5 mM glucose. The ratio of insulin secretion to amylase secretion in the culture medium increased with the advance of culture days although secretions of both insulin and amylase decreased individually. On the 7th culture day, short term incubations were performed to test with various insulin secretagogues; obvious insulin release into the incubation medium was observed. These results show that the pancreatic chamber also in vitro secretes insulin rapidly and significantly in response to various stimuli; that by longer culture of a neonatal rat pancreas in this device, insulin secretory cells without exocrine tissue would be obtained without using digestive enzymes; that application of a pancreatic chamber for a pancreatic transplantation may be feasible.     

Use of plastics for suspension culture vessels

Summary Plastic containers were found to facilitate the adaptation of a number of mammalian cell lines derived from normal tissues to growth in continuous suspension culture. Several different types of plastic materials were studied and a variety of culture vessels were designed.     

Human mammary cancer cell lines and other epithelial cells cultured as organoid tissue in lenticular pouches of reinforced collagen membranes

Summary A system has been developed for the culture of cells that provides conditions favoring the formation of tissues comparable to conditions existing in nature. The culture chamber is a lens-shaped pouch composed of two thin-walled, reinforced, waffled collagen membranes facing each other. The chamber is immersed in immersed in medium in a closed transparent container and incubated on a rocker. On histologic study, after days to weeks in culture, human mammary cancer cell lines BT-20, MCF-7, MDA-231, MDA-468, and T47D grow in the chamber as distinctive structured epithelial tissue. Dog kidney cell line MDCK grows as a papillary adenocarcinoma and rat bladder cancer line NBT-II as an epidermoid carcinoma; cells from clinical effusion tumors produce distinct tissue. Changes in histologic phenotype may be driven by molecular changes at the level of the genome. Resulting alteration of the biochemical functions essential for the integrity of specific durable tissue organization should alter or reset the pattern of tissue organization and of biological behavior, including malignancy and response to cytotoxic chemicals. Lenticular pouch culture promises to be an effective tool for exploring the molecular changes associated with histogenesis and malignancy. This paper is dedicated to the memory of Clyde J. Dawe, who died suddenly on Cape Cod, Massachusetts, in a glider accident on July 5, 1996. We were friends for about 40 years. Clyde was one of the finest scholars in cancer biology I have ever known. I learned many things from him and miss him.     

Reemergence of covert bacteria Bacillus pumilus and Brevibacillus sp. in microbe-freed grape and watermelon stocks attributable to occasional autoclaving-defying residual spores from previous cycles

Covert bacteria-harboring long-term micropropagated culture of grape that was sanitized of associated microbes as assessed through the indexing of tissue and medium for several passages (Thomas and Prakash (2004) In Vitro – Plant 40:603–607) showed reemergence of bacteria in a section of cultures (3/40) during the subsequent screening. Similarly, long-term propagated triploid watermelon cultures showed bacterial reentry in some sub-stocks (2/14) during their extended monitoring post-antibiotic treatment (Thomas et al. (2006) Plant Cell Tiss Org Cult, 85:317–329). Each of the three grape sub-stocks showed single bacterium, identified as Bacillus pumilus (×2) or Brevibacillus sp. (×1) based on 16S rDNA sequence analysis with 100% similarity to ‘ARBG2’ and ‘ARBG1’ isolates respectively, retrieved originally from grape stocks as long-term alcohol-surviving spore-forming covert contaminants. The two isolates from watermelon included Brevibacillus sp. (ARBG1) and B. pumilus (AuRB-WM1), the latter distinct from the ‘ARBG2’ isolate. The chance of bacterial re-entry through endophytic survival or during culture handling appeared remote on account of the tissue indexing employed and the care taken. Monitoring the autoclaves for proper functioning using chemical, physical and biological indicators, sealing the vessels to protect from microscopic vectors and scrutinizing the autoclaved medium for microbial growth all pointed the contaminant source to occasional vessel-adhering residual spores from the previous covertly contaminated cycle(s) that were not recognized as contaminated. Such spores withstood the standard autoclaving either due to their high heat resistance or some conditions typical to tissue culture vessels, and the proportion of vessels showing carry-over inoculum increased with the number of passages of culturing with the organism. The problem due to these hardy spore-forming bacteria was circumvented through double autoclaving of culture vessels with the first one a day prior to the medium preparation     

Simulated microgravity culture system for a 3-D carcinoma tissue model

An in vitro organotypic culture model is needed to understand the complexities of carcinoma tissue consisting of carcinoma cells, stromal cells, and extracellular matrices. We developed a new in vitro model of carcinoma tissue using a rotary cell culture system with four disposable vessels (RCCS-4D) that provides a simulated microgravity condition. Solid collagen gels containing human pancreatic carcinoma NOR-P1 cells and fibroblasts or minced human pancreatic carcinoma tissue were cultured under a simulated microgravity condition or a static Ig condition for seven days. NOR-P1 cultures subjected to the simulated microgravity condition showed greater numbers of mitotic, cycling (Ki-67-positive), nuclear factor-kappa B-activating cells, and a lower number of apoptotic cells than were shown by cultures subjected to the static Ig condition. In addition, human pancreatic carcinoma specimens cultured under the simulated microgravity condition maintained the heterogeneous composition and cellular activity (determined by the cycling cell ratio and mitotic index) of the original carcinoma tissue better than static culture conditions. This new 3-D rotary cell culture system with four disposal vessels may be useful for in vitro studies of complex pancreatic carcinoma tissue.     

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth. © 1993 Wiley-Liss, Inc.     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering

Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).     

向日葵Ti T—DNA转化系的原生质体培养和细胞培养

根癌农杆菌离体感染向日葵子叶、下胚轴外植体形成的Ti T-DNA转化组织在激素条件下长期继代培养后,用来进行原生质体培养和细胞培养。适于B6S3转化系和T37转化系原生质体培养的培养基分别为附加不同激素和糖类的C81V和DPD培养基。用液体浅层法培养3～5天时,原生质体开始分裂。10天后形成细胞团。B6S3转化系还可直接从原生质体产生原胚状结构。转化系的细胞克隆均保持着激素自主型生长特性和冠瘿碱合成酶合成特性。     

Low-energy X-radiation for prevention of restenosis results in localized inhibition of V79 fibroblast cell proliferation

Local delivery of high-energy ionizing radiation by using 3 or 3 emitters to injured vessels demonstrated inhibition of cell proliferation (CP) and neointima formation. Low-energy ('soft') X-radiation (LEXR) offers logistic and safety advantages over the use of disposable radioisotopes. This study evaluated the efficacy of LEXR in penetration and inhibition of CP at doses similar to those prescribed for the use of radioisotopes for prevention of restenosis. Serial measurements in an ion chamber detected the attenuation of LEXR using potentials of 17 and 40 kV at a distance of 17 cm of air through 0-10 mm depths of serum-containing tissue culture medium. The effect of inhibition on CP was determined by exposing V79 fibroblasts to a potential of 17 kV in order to deliver a prescribed dose of 13 Gy at a dose rate of 2.17 Gy/min to the surface of the cells. Complete inhibition of CP at a height of 0.00 mm occurred with 13 Gy; however, a 50% attenuation of the dose was measured at a medium depth of 1.22 mm and was associated with a reduction of 60% of the CP. LEXR demonstrated an ability to inhibit CP at doses equivalent to those used in techniques involving 3 and 3 irradiation. Under such conditions, the dose gradient is too high, especially for large vessels. However, a catheter-based LEXR that could be inserted into the artery with the capability of varying effective energy would be ideal for intravascular applications.     

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia)

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.     

Neutral red uptake assay for the estimation of cell viability/cytotoxicity

The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.     

The use of a conventional tissue culture plate as an optically accessible perfusion chamber for in situ assays and for long-term cultivation of mammalian cells

An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 10² cells cm^-2 for the adherent cells and 5 × 10⁴ cells mL^-1 for the suspension cells. The upper limits are 1–2 × 10⁵ cells cm^-2 respectively 8 × 10⁶ cells mL^-1.     

Glucose and amino acid metabolism in an established line of skeletal muscle cells

The suitability of an established myogenic line (L₆) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days at which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized at a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate at an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10–24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose-derived pyruvate enters mitochondrial pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.     

Use of a tissue culture chamber for developmental studies of aquatic phycomycetes

Summary A mammalian tissue culture chamber was utilized in a developmental study of two aquatic fungi. This apparatus was found to provide a simple system by which aquatic microorganisms could be continuously cultured and their development directly observed under precisely controlled environmental conditions.     

Interactions of culture vessels, media volume, culture density, and carbon dioxide levels on lettuce and spearmint shoot growth in vitro

 The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 μmol mol^–1 CO₂ after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 μmol mol^–1 CO₂. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an elevated CO₂ environment or larger culture vessels. Received: 22 December 1998 / Revision received: 2 July 1999 / Accepted: 12 July 1999     

Evaluation of Gentamicin for Use in Virology and Tissue Culture

Data are presented comparing gentamicin to penicillin and streptomycin (Pen-Strep) in tissue culture medium with respect to a number of parameters associated with virology and tissue culture. Unlike Pen-Strep, gentamicin was stable at pH 2 to 10 for 15 days at 37 C in tissue culture medium, and its activity was unaffected by the presence of serum. Moreover, it was stable to autoclaving. Twenty cell types replicated normally at the suggested concentration of 50 mug/ml, and all cells were unaffected by 20 times this concentration. Evidence for its practical use in virus studies was demonstrated in that (i) it was not viricidal to ribonucleic acid or deoxyribonucleic acid viruses at 40 times the suggested concentration at 37 C, (ii) the size and number of plaques were not affected by 20 times the suggested concentration, (iii) interferon assays and production were unaffected by 20 times the suggested concentrations. Gentamicin may be uniquely useful for shipment of clinical specimens and long-term tissue culture and virus studies.     

A programmable micropropagation apparatus using cycled liquid medium

An apparatus was developed that featured programmable application of liquid medium to plant cultures for micropropagation. Computer control capabilities included liquid medium introduction and medium depth within four culture vessels, medium application and removal on an assigned schedule, schedule adjustment during a culture period and medium replacement. The medium level was controlled using an accurate custom level-sensing technique consisting of thermistors and float switches. Seven-liter polycarbonate containers were modified and used as the culture vessels. Maintaining sterility was a key constraint in the development of the plant tissue culture apparatus.     

Evaluation of cell culture flasks designed for experiment under altered gravity-vector conditions.

Cell culture flasks applicable for altered gravity conditions, such as centrifugation, clino-rotation or microgravity in space, were manufactured for trial. The flask has flat polystyrene surface for monolayer culture and gas-permeable film window on the opposite face. The space in-between consists the culture chamber to be filled with liquid medium. To reduce the water loss and bubble formation in the culture fluid, another gas permeable window was placed on top to form a space where distilled water may be filled. The double-decker culture flask can be used for both space and ground-based experiments in common.