Cells In Petri Dish Research Articles

The Quick Antigen Identification By Tissue Flow Cytometry for CAR-T Therapy in Relapsed/Refractory B-Cell Malignancies without Bone Marrow Involvement or Serous Cavity Effusions

In recent years, chimeric antigen receptor (CAR) T-cell therapy has been widely used to treat relapsed/refractory B-cell malignancies. Before CAR-T, the identification of target antigens on tumor cells is needed for choosing a suitable antigen-specific CAR, which is usually performed by multiparameter flow cytometry (FCM) for bone marrow (BM) samples or immunohistochemical (IHC) staining for tissue samples. However, some patients with tumor masses only and without BM involvement or serous cavity effusions (SCE) may not have much time to wait the results from IHC staining owing to rapidly growing tumor cells, therefore, we established a quick antigen identification by FCM using tissue samples. Fresh tumor tissues taken by fine or coarse needle puncture from different body parts were put in saline with 2% fetal calf serum and sent to the FCM lab as quickly as possible, once in lab, samples were processed immediately. Tissue strips were placed into a 60 mm petri dish, gently pressed by a syringe piston and repeatedly flushed with saline to separate cells from tissues; then the liquid with cells in petri dish were transferred into a 100μm cell strainer on top of 50 mL conical tube to exclude non-cellular tissue components, the strainer was washed with saline for 2-3 times. The strained cell suspension in conical tube was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded by pipetting, cells were counted and resuspended with saline at a concentration of 104-105/ml, then the single cell suspension was ready for flow cytometry assay. 7-AAD and antibodies CD19/CD22/CD7/CD45 were added into the first tube to quantify viable cells and identify cells available for analysis, if dead cells were more than 30% of all cells, 7-AAD was needed for all next tubes, otherwise, it could be omitted. The standard antibody panels consist of CD10/CD20/CD34/CD19/CD38/CD45 for precursor B-cells and kappa/lambda/CD10/CD19/CD20/CD38/CD45 for mature B-cells, extra antibodies could be added according to patient's situations. Samples were performed on the 8-color BD FACSCanto II flow cytometer, and analyzed by BD FACS Diva software or Kaluza software from Beckman Coulter. Antigen expression, partial expression and no expression were defined as >80%, 20-80% and <20% of gated cells displaying interested antigen, respectively. Dim expression was defined as more than one log weaker than the normal counterpart's mean fluorescence intensity (MFI). From October 2017 through June 2020, a total of 63 patients with relapsed/refractory B-cell malignancies who had tumor masses only and no BM involvement or malignant SCE were hospitalized in our center for CAR-T treatment after failure of multiple-line therapies, including 11 (17.5%) acute lymphoblastic leukemia (ALL) and 52 (82.5%) non-Hodgkin's lymphoma (NHL), the median age was 41 years (range, 1-71) with both adults (n=47, 74.6%) and children younger than 18 years (n=16, 25.4%). Acquired events from tissue samples varied from 1000 to more than 100, 000 each tube (5 cases <5000 and 17 cases >100,000), the percentages of tumor cells in all cells available for analysis varied from 0.03% to 99.44% (5 cases <5% and 13 cases >90%). We have CD19-, CD20- and CD22-specific CAR-T cells for B-ALL and B-NHL, the choice of a CAR-T product depends on the antigen expression on tumor cells, patients with partial or no or dim expression (PND expression) of certain antigen are not suitable for the relevant antigen-specific CAR-T therapy. Among this cohort of patients, CD19 expressed in all but 1 ALL case who had accepted CD19 CAR-T; PND expression of CD20 occurred in 19.2% (10/52) of NHL patients since they were repeatedly administrated anti-CD20 antibodies in previous treatments which resulted in antigen diminishment or loss, all B-ALL patients had no or partial CD20 expression; PND expression of CD22 was seen in 18.2% (2/11) of ALL and 13.5% (7/52) of NHL. All flow results came back within hours (< 4-6 hours), much quicker than results from IHC staining which takes days. In conclusion, the correct selection of a target antigen is the basis of effective CAR-T treatment in relapsed/refractory B-cell malignancies especially for patients having accepted antibody or CAR-T therapies, our antigen detection by tissue flow cytometry provided a quick and accurate antigen identification for patients with no BM or SCE samples. Disclosures No relevant conflicts of interest to declare.

Magnetic Patterning of Vorticella convallaria in a Microfluidic Device.

We describe an inexpensive magnetic cell patterning method as a tool for protozoologists. The ciliate Vorticella convallaria is useful for various biofluidics applications. Here, we show that V.convallaria will ingest metal beads and that permanent magnets can be used to pattern cells in Petri dishes or a microfluidic device. Patterning is reversibly achieved by placing magnets at the point of desired cell attachment. Analogous magnetic manipulation could be performed using other phagocytic cells.

A Wideband Microwave Exposure Setup for Suspended Cells Cultures: Numerical and Experimental EM Characterization

An in vitro exposure setup developed to expose suspended cells in Petri dishes on a large frequency band up to a few gigahertz was studied. This system was based on far-field exposure with a horn antenna in an anechoic chamber. A specific incubator was designed with a water-cooling system for constant temperature inside the setup. Four exposed Petri dishes presented a similar specific absorption rate (SAR) distribution with a whole-volume SAR of $0.56 \pm 0.40~\hbox{W/kg/W}_{\rm inc}$ at 2.45 GHz. The incubator was equipped with a stirring mechanism allowing an homogenous distribution of temperature in the biological solution. The SAR was assessed over the 1.5–3 GHz frequency range. Using the temperature control and stirring, an increase of 0.4 $^\circ {\rm C}$ in temperature was observed after 2 h exposure at 2.45 GHz, 22 W incident power. This dosimetric study demonstrated the ability of this system to expose cells in suspension at different frequencies in a controlled environment .

Chromatin Immunoprecipitation (ChIP) using <em>Drosophila</em> tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest. ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay. Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo(1, 2). Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

Optimizing stem cell culture

Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane.

A Shortcut to Immortality: Rapid Reprogramming with Tissue Cells

A Shortcut to Immortality: Rapid Reprogramming with Tissue Cells

The Selection of Serum-independent PC12 Cells for a More-reliable Manganese Cytotoxicity Test

A major issue concerning the protocols of heavy metal cytotoxicity tests with PC12 cells was the hypothesis that serum in the culture medium might sequester the metal, thus altering the results obtained. However, serum withdrawal impairs the viability of PC12 cells themselves, thus impeding cytotoxicity testing in the absence of serum. In this study, we repeatedly selected undifferentiated, totally non-adherent PC12 cells in Petri dishes. Surprisingly, we discovered that these cells could survive and proliferate in serum-free medium. Moreover, features such as NGF-responsiveness, resazurin reduction potential, doubling rate, protein content, and basal caspase-3 enzyme activity, were equivalent to those exhibited by standard PC12 cultures. Further experiments aimed at fully characterising these serum-independent PC12 cells are in progress. These cells enabled cytotoxicity experiments to be conducted with manganese, both in serum-supplemented and in serum-deprived medium. The results demonstrated that serum removal decreased the LC50 of manganese from 250microM to 32microM, without affecting the internalisation of the metal. The data exclude an early competitive effect of serum on metal internalisation; rather, they suggest a late protective mechanism mediated by serum against the cytotoxic effect of the already-internalised metal.

Perifusion culture of hepatocytes.

AbstractCultivation of cells in Petri dishes or culture flasks is usually performed by changing the medium after certain periods of time, e.g., 24 h (stationary cultivation). Clearly, the concentration of nutrients and other components as well as of added hormones will decrease over this period because of the metabolic activity of the cultured cells Conversely, metabolic end products such as lactate, ammonium ions and even bile salts will accumulate. In order to replace the sequential changes of the culture medium by a continuous supply, several systems for perifusion or circumfusion of the cells with medium have been designed. Usage of one such approach, reported for cultivation of hepatocytes (1), indicated that perifusion was beneficial to the cultured hepatocytes, resulting in a prolonged life-span and an enhanced metabolic performance In particular, it was found that the hormonal response to dexamethasone and glucagon was increased (2), partly because steady-state concentrations of hormones are established even at low initial concentrations. Since these early studies, the technique for perifusion has improved and it has been found that oxygen tension within the culture medium is a very critical parameter; surprismgly, in contrast to previous assumptions that oxygen supply in normal stationary cultures is insufficient (3), higher oxygen tensions, in conjunction with elevated flow rates, proved to be cytotoxic under perifusion conditions. If this and other special features and requirements are considered, perifusion cultivation has considerable advantages over conventional culture techniques, particularly for studies on biotransformation and drug toxicity (4, 5).KeywordsCulture ChamberNewborn Calf SerumCulture SupportMedium ReservoirBubble TrapThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion

The classical mode of luteinizing hormone (LH) secretory desensitization in the rat appears after 3–6 h of continuous in vitro administration of gonadotropin (GnRH). A second mode has been reported to occur very rapidly (<2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal. Here, the existence of a third mode of desensitization is reported, occurring at 40–50 min after initiation of continuous GnRH administration. Rat pituitary cells were perifused with 10 −8 M GnRH for 6 h; 10 min samples were collected for LH measurements by radioimmunoassay. As expected, the pattern of LH release was biphasic: LH levels peaked in the first phase at 30 min, decreased at 40–50 min, increased in the second phase to maximal levels at 90–110 min, and then decreased in the classical desensitization mode to near-baseline values by 300–360 min. Static incubations of pituitary cells in Petri dishes in the presence of high (10 −8 M) or submaximal (10 −9 M) GnRH concentrations confirmed the decrease in LH secretion at 40–50 min. Measurement of LH by reverse hemolytic plaque assay (RHPA) confirmed the existence of this new mode of desensitization; since 93% of all gonadotropes had become secretory at 40–50 min, the possibility of two subpopulations of gonadotropes accounting for the two phases of LH secretion appears to be ruled-out. GnRH receptor binding studies demonstrated a ∼50% decrease in cell-surface binding in association with the desensitization at 40–50 min. These studies suggest the existence of a third mode of GnRH-induced LH secretory desensitization that is not due to gonadotrope subpopulations but may be causally associated with decreased GnRH receptor binding.

A new, optical-lever based atomic force microscope

A new optical-lever based atomic force microscope is described in which the cantilever scans and is accurately tracked by a scanning focused spot. It can operate at forces below one nanoNewton over image areas greater than 100 μ×100 μ. It can be combined with optical microscopes of high numerical aperture and operated with the sample and cantilever in fluids. As examples of its applications, images of living cells in Petri dishes and a 6 in. (15.24 mm) silicon wafer are included.

Volatile anesthetics inhibit the ion flux through Ca 2+-activated K + channels of rat glioma C6 cells

Ca 2+-activated K + channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes. The ion flux through the channels was studied with 86Rb + after addition of a Ca 2+-ionophore to the incubation medium. Both the influx and efflux of 86Rb + through these Ca 2+-activated K + channels were inhibited by the general anesthetic halothane (at clinical concentrations). Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca 2+-activated K + channels at clinical concentrations. Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.

Homogeneous growth of tumor cell colonies in agar containing glas capillaries

The capillary cloning system has been shown to have advantages over conventional cloning of human tumor cells in Petri dishes. In the present study a further optimization towards homogeneous colony distribution and high cloning efficiency is described. For reasons of reproducibility the study focused on cell lines, i.e. three human linew (MDA-231, HT-29, L363) and one rodent line (CHO-AB). Major variables investigated were the gel length, the capillary tube diameter, the tube sealing and buffer system, and the cell number. Criteria for optimal tumor colony growth included homogeneous colony distribution along the gel, mean colony size and cloning efficiency. It was found that colony distribution as well as overall colony growth depended largely on the gel length, i.e. on the volume of tumor cell containing agar applied per capillary tube. The results showed that optimal tumor cell colony growth was achieved in 100 ul capillary tubes of 1.2 mm internal diameter filled with 30ul, yielding a gel length of 27 mm. Colony formation did not significantly differ between sealed and unsealed tubes, provided that HEPES buffer was added. It was concluded that, for practical reasons, sealing of tube ends and therefore utilization of HEPES buffer is not necessary. In a head to head comparison, cloning efficiency was equal or higher in capillary tubes than in Petri dishes. The capillary cloning system is an alternative for drug development as well as for predictive drug testing. Its major advantage is the utilization of fewer tumor cells.

An automatic counter for autoradiographed cells in Petri dishes.

A simple automatic counting device for cells on Petri dishes is described. The device is tested up to 1 x 10(4) cells (0.01 m)-2 and counts linearly without coincidence error. The counting time for each dish is c. 3-4 min including the time required to place the dish in the apparatus.

Attempts to produce in vitro the reaction of immunologically competent cells of C57BL/6 origin to Balb/c embryo fibroblasts

AbstractSpleen or lymph node cells of C57BL/6 origin or both were mixed with subcellular material from Balb/c tissue for several hours at 37°C. The subcellular material was then washed out (by repeated centrifugation) with PBS and the immune cells were placed either in diffusion chambers in the peritoneal cavity of Swiss mice or in Petri dishes with C57BL/6 embryo cell monolayer (feeder layer) and incubated in a thermostat at an atmosphere of 5% CO2. After 4 to 5 days these cells were added to target Balb/c embryo cells in Petri dishes. The first control was done by sensitiziing the cells with syngeneic material, the second control by adding the immune cells to syngeneic target embryo cells. The sensitized cells mixed with allogeneic embryo cells produced a positive reaction in the form of agglutination around the target cells and destruction of these cells. In controls the immune cells were seldom found and were randomly distributed.

A Simplified Tissue-Culture Technique to Detect Rous Sarcoma Virus and Its Application in Antibody Assays

It has been known for several years that chickens from most flocks have varying levels of Rous sarcoma virus (RSV) neutralizing antibodies (3). Although RSV is not found in the general poultry population, most, if not all, avian tumor viruses are antigenically related (1) and it is presumed that the RSV antibodies are actually induced by leukosis virus. It is therefore understandable that there is considerable interest in the occurrence of these antibodies and a need for means of assaying them. Modifying a tissue-culture technique described by Manaker and Giroup& (4) for demonstrating RSV, Temin and Rubin developed an assay system for titrating virus (8). Chick embryo fibroblasts infected with RSV transform into Rous sarcoma cells. These transformed, or altered, cells multiply and, after several days, produce discrete, microscopic foci that can be counted for estimations of virus titer. For an antibody test, Rubin (7) inoculated Petri-dish cell cultures with virus alone or with virus-serum mixtures and then later counted the resultant foci. A reduction of 50% or more in the number of foci below that of controls indicated the presence of antibodies in the serum. This neutralization test appears to be quite reliable and gives consistent results as long as very exacting conditions are