Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.     

THE CIRCUMFUSION SYSTEM FOR MULTIPURPOSE CULTURE CHAMBERS : II. The Protracted Maintenance of Differentiation of Fetal and Newborn Mouse Liver in Vitro

The circumfusion system is a complex in vitro pumping unit incorporating 12 multipurpose culture chambers through which a serum-supplemented fluid nutrient is recirculated at a rate of 4.5 ml/min per chamber. This system was used to study the differentiative responses of fetal and newborn mouse liver explants placed in the serum-free environment formed between the sheets of unperforated cellophane and cover glasses of the chambers. Hepatocytes (parenchymal cells) were discernible in 3–5 days. They retained many of their features of differentiation in the circumfusion system for more than 120 days of cultivation. The living morphological characteristics of the hepatocytes were studied by phase-contrast microscopy (direct viewing and time-lapse cinemicrography) and by special cytochemical staining. Electron micrographs were made of both fresh liver specimens and the cultured cells. Comparisons of the cultured parenchymal cells with their in vivo progenitors showed a remarkable preservation of their differentiated state.     

A Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

Microscopy with Plastic Substitutes for Cover Glasses

To determine whether plastic substitutes for cover glasses on microscope slides affect the performance of the microscope, their optical constants were determined. The plastic covers and glass cover glasses were mounted also on silvered slides to form Star Test Plates which were studied by competent observers. The thicker plastic cover glasses, now on the market, are satisfactory when mounted to give a plane surface. Optically inhomogeneous materials, of irregular thickness, those that curl or do not have plane surfaces, adversely affect the performance of the microscope and should not be used. Since the substitutes are softer than glass they must be protected from abrasion. It is recommended that thicknesses of 0.18 mm., and none outside of a range of 0.12 to 0.20 mm. be used. For critical observation with unimmersed objectives of high aperture, best results are obtained with the correction collar set at the position corresponding to the actual thickness of the cover slip, just as would be done with glass cover glasses.     

A Plexiglas Isolation-Transfer Chamber for Use with the Fonbrune Micromanipulator

The chamber is made with two pieces of clear Plexiglas, both 1 inch wide and 3 inches long; the top piece, 1/16 inch thick; the bottom, 1/4 inch. A recess 14 mm wide, 37 mm long and 5 mm deep is cut into the bottom piece leaving a margin 1.5 mm wide, this margin is then cut down to a height of 2.5 mm for the length of the recess; a piece of moistened filter paper I1/2 inches long and 5 mm deep is attached to the rear wall, leaving the bottom clear for the transmitted light; two notches 13 × 15 mm and 12 mm apart are cut from the top piece. The top is superimposed on the bottom and held by two screws to form a chamber that is accessible to microdissection instruments through its open side. Two cover glasses, one bearing the specimen and the other, the medium to which the transfer is to be made, are placed over the notches in the top (agar side down). The actual transfer of material is made by shifting the position of the cover glasses with the mechanical stage of the microscope while the specimen is held by the micromanipulator.     

Relationships between Presence of the Eye Cup and Maintenance of Lens-Forming Capacity in Larval Xenopus laevis

The lentectomized eye of larval Xenopus laevis can regenerate a lens by a process of lens-transdifferentiation of the cornea and pericorneal epidermis. These tissues can form the lens only when they become in direct communication with the environment of the vitreous chamber (neural retina) indicating that the eye cup plays a fundamental role in this process.
In this work the role of the eye cup in the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis was studied by allowing these tissues to cover the enucleated orbit for different periods, and then implanting them into the vitreous chamber of the contralateral eye. Under these experimental conditions the maintainance of the lens-forming capacity of the cornea and pericorneal epidermis showed no significant correlation with the time from enucleation to implantation.     

Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system

 When cultured on a polystyrene surface or aminoalkylsilane-coated cover glasses, rat and human hepatic stellate cells exhibit a flattened, fibroblast-like shape with well-developed stress fibers. However, culturing the cells on type I collagen gel results in the elongation of long, multipolar cellular processes, whereas cells cultured on Matrigel maintain their round shapes. Dual fluorescence staining of microtubules and fibrillar actin indicated that the processes extend together with collagen fibers and contained microtubules as the core, whereas the periphery contained fibrillar actin. Immunofluorescence staining of vinculin showed that the focal adhesions were distributed mainly in lamellipodia when cultured on aminoalkylsilane-coated cover glasses, whereas in the cells cultured on type I collagen gel they were localized to the tips of the processes and along their bottom surface contacting collagen fibers. Wortmannin, as well as staurosporin and herbimycin A, inhibited the elongation process and induced the retraction of elongated processes. The wortmannin treatment also resulted in an alteration in focal adhesion distribution from the processes to cell bodies. These results indicate that the cell surface integrin binding to interstitial collagen fibers induces the elongation of processes through signaling events and the subsequent cytoskeleton assembly in hepatic stellate cells. Accepted: 12 February 1998     

Assessment of mitochondrial function in the surviving cytoplasts of cultured cells of pig embryonic kidney by using ethylrhodamine

A study was made of the functional state of chondriome in cytoplasts previously cultivated for a long time under vital staining with fluorescent dye ethylrhodamine (10 micrograms/ml) which is known to accumulate preferentially in mitochondria. The energization was estimated by the intensity of mitochondrial fluorescence. It was realized that 30 minutes after enucleation cytoplasts retained the same fluorescence as did the untreated cells, and that the mitochondrial distribution within the cell was very similar. Such a high intensity of fluorescence seen within one day after enucleation gives a strong evidence on the high degree of independence on the cell nucleus of organelles that provide the energy to metabolic processes. In the course of survival in the cultivation medium for 1-4 days the intensity of fluorescence is shown to fall, especially on the second day after enucleation. All these changes coincide with the changes in cytoplast shape: originally well spread bodies transform into squeezed, ball-shaped or strongly deformed ones. The adhesive ability is going down, and in result only single units of cytoplast can hardly be found on the cover slips by the 4th day after enucleation. These changes give evidence on the enhanced degeneration of cultured cytoplasts.     

A flow system for the study of shear forces upon cultured endothelial cells

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.     

A method to generate microcells from human lymphoblasts for use in microcell mediated chromosome transfer

Summary A method is described to generate microcells from human lymphobalsts for use in microcell-mediated chromosome transfer (MMCT). Micronuclei were induced in cells from a human lymphoblastic cell line by prolonged colcemid treatment, and were separated from these lymphoblasts by: (a) attaching the cells to Concanvalin A coated plastic slides designed for enucleation, and (b) centrifuging the slides in medium containing cytochalasin B. Microcells of less than 3 μm in diameter were fused with thymidine kinase negative mouse fibroblast (LMTK⁻). HAT medium (hypoxanthine, aminopterin and thymidine) was used to select microcell hybrids expressing thymidine kinase activity. Positive clones were isolated and Q-banded for chromosome analysis. Unlike previous methods this procedure permits microcells to be easily generated from lymphoid cells. This methodology of enucleation of microcells may be extended to a variety of other donor cell types which can be micronucleated but which do not adhere tightly to enucleation slides and do not exhibit extrusion subdivision. This feature makes our methodology particularly useful for constructing a library of hybrid clones containing one or a few human chromosomes.     

Electron microscopy of free-floating cells: thin-layering technique, and selection of individual cells for ultramicrotomy.

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily be relocated by the aid of the pre-embedding reference micrographs.     

Electron Microscopy of Free-Floating Cells: Thin-Layering Technique, and Selection of Individual Cells for Ultramicrotomy

A rapid and accurate procedure for electron microscopy of individual cells from suspensions (blood, peritoneal exudate, etc.) is described. After fixation of the sample with standard techniques, the particulate constituents are suspended in buffered 5% bovine serum albumin, thin-layered by gravity on clear supports (cover glasses or polyester slips) in which an orientation grid had been scored, and then immobilized by exposure of the preparations to acrolein vapors. The specimens are examined for cells of interest under a light microscope using interference or phase contrast; individual cells to be sectioned are documented in three photomicrographs taken at different magnifications. After this the specimens are embedded like ordinary cover slip preparations. When examining the face of the polymerized block under a light microscope, the position of the selected cell beneath the orientation grid relief can readily he relocated by the aid of the pre-embedding reference micrographs.     

A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

A Staining Rack for Handling Cover-Glass Preparations

A porcelain staining rack (Fig. 1) has been devised for handling cover-glass preparations. The design is along the general lines of staining racks for slides commonly sold by commercial houses. It consists of three parallel rods—one at the bottom, two on the sides. These three rods are held together by two end pieces. Each of the rods has twelve slots, thus the rack holds twelve cover glasses. The slots have been arranged to hold the cover glasses some distance apart. Circular cover glasses having a diameter of 22 mm. are most desirable. Each of the two end pieces has a hole near the top for inserting one end of the wire tongs or handle (see Fig. 1) which is used for removing the rack from the stender dish. The staining rack is 40 mm. long and 35 mm. high and is designed, as shown in the figure, to fit the ordinary Stender dishes commonly used in many laboratories.     

A Simple Permanent Aceto-Orcein Stain for Free Cells, Cultures and Homogenates

A simplified technic for preparation of the aceto-orcein stain permits the storage of cells in the stain or squash preparations at room temperature for long periods without in* jury to or distortion of the cells and mitotic plates. Fresh cells from tumor ascites, tissue culture cells growing in free suspension or over cover slips, and homogenates of whole tissues are stained directly in a test tube in either (1) regular aceto-orcein and subsequently mounted in glycerol, or (2) aceto-orcein-glycerol mixture. These preparations are squashed for chromosome counts, and the permanent slides are kept from drying out by ringing the cover slip with Damar or Permount.     

A technique for in situ karyotyping of primary amniotic fluid cell cultures.

A time proven technique is described for growing amniotic fluid cell cultures on cover glasses in Leighton tubes and for processing the mitotic cells in situ. Karyotyping the clones in situ eliminates most of the problems caused by somatic chromosome mutations in vitro and by maternal cell growth.     

Induction of measles virus hemagglutinin in a persistently infected, nonvirogenic line of cells (BGM/MV).

BGM/MV cells carry measles virus antigens and nucleocapsid-like structures in their cytoplasm. There is no infectious virus demonstrable, and measles virus-induced cell surface changes detectable by hemadsorption (HAD) are absent. Treatment of cells with actinomycin D or cycloheximide or enucleation of cells with cytochalasin B induced surface changes in that the cells became HAD positive. 6-Azauridine treatment of cells did not inhibit the induction of HAD, suggesting that RNA synthesis was not required. Cycloheximide treatment of cells induced by enucleation inhibited the development of HAD, suggesting a requirement for protein synthesis.     

An improved assay to quantitate the invasiveness of cells in modified Boyden chambers

The most commonly used means of assessing the invasiveness of cultured cells is the Boyden chamber assay, which requires that cells lyse Matrigel™, followed by migration through pores in a filter in response to a chemotactic gradient. This report describes a simple method, which greatly increases the speed and accuracy by which Boyden chamber assays can be analyzed, and permits the concurrent analysis of distinct cell subpopulations within specimens containing multiple-cell types.     

Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts

We have used murine splenic erythrolasts infected with the anemia- inducing strain of Friend virus (FVA cells), as an in vitro model to study cytoskeletal elements during erythroid maturation and enucleation. FVA cells are capable of enucleating in suspension culture in vitro, indicating that associations with an extracellular matrix or accessory cells are not required for enucleation to occur. The morphology of FVA cells undergoing enucleation is nearly identical to erythroblasts enucleating in vivo. The nucleus is segregated to one side of the cell and then appears to be pinched off resulting in an extruded nucleus and reticulocyte. The extruded nucleus is surrounded by an intact plasma membrane and has little cytoplasm associated with it. Newly formed reticulocytes have an irregular shape, are vacuolated and contain all cytoplasmic organelles. The spatial distribution of several cytoskeletal proteins was examined during the maturation process. Spectrin was found associated with the plasma membrane of FVA cells at all stages of maturation but was segregated entirely to the incipient reticulocyte during enucleation. Microtubules formed cages around nuclei in immature FVA cells and were found primarily in the incipient reticulocyte in cells undergoing enucleation. Reticulocytes occasionally contained microtubules, but a generalized diffuse distribution of tubulin was more common. Vimentin could not be detected at any time in FVA cell maturation. Filamentous actin (F-actin) had a patchy distribution at the cell surface in the most immature erythroblasts, but F-actin bundles could be detected as the cells matured. F-actin was found concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts. Newly formed reticulocytes exhibited punctate actin fluorescence whereas extruded nuclei lacked F-actin. Addition of colchicine, vinblastine, or taxol to cultures of FVA cells did not affect enucleation. In contrast, cytochalasin D caused a complete inhibition of enucleation that could be reversed by washing out the cytochalasin D. These results demonstrate that F-actin plays a role in enucleation while the complete absence of microtubules or excessive numbers of polymerized microtubules do not affect enucleation.