The biophysical aspects of reconstructing a single cell by the methods of cell engineering

The problem of the low efficiency of mammalian cloning is discussed with emphasis on the need of expert assessment of every step in single cell reconstruction, beginning with microsurgical manipulations. Experimental proof is provided for the impairment of cell integrity upon its fixation for microsurgery by the negative pressure in a conventional holding pipette. The ensuing leakage of the cell contents is shown to depend on the value of negative pressure, the duration of holding, and the size of the holder orifice. An alternative method of cell fixation is proposed, taking advantage of the capillary forces in the holding micropipette. This reduces the holding effort by two orders of magnitude and raises the cell survival upon microsurgery at least to 92%. To alleviate cell damage by instrumental invasion, a new technique is proposed for making micropipettes. Another novel method is offered for pipette filling with viscous liquid such as DNA solution, which allows continuous injection of more than 1000 cells.     

Viscoelastic properties of transformed cells: role in tumor cell progression and metastasis formation

The micropipette aspiration technique was used to investigate the deformation properties of a panel of nontransformed and transformed rat fibroblasts derived from the same normal cell line. In this method, a step negative pressure is applied to the cell via a micropipette and the aspiration distance into the pipette as a function of time is determined using video techniques. A standard solid viscoelastic model was then used to analyze the viscoelastic properties of the cell. From these results, it is concluded that a direct correlation exists between an increase in deformability and progression of the transformed phenotype from a nontumorigenic cell line into a tumorigenic, metastatic cell line.     

Micropipette aspiration on the outer hair cell lateral wall

The mechanical properties of the lateral wall of the guinea pig cochlear outer hair cell were studied using the micropipette aspiration technique. A fire-polished micropipette with an inner diameter of approximately 4 microm was brought into contact with the lateral wall and negative pressure was applied. The resulting deformation of the lateral wall was recorded on videotape and subjected to morphometric analysis. The relation between the length of the aspirated portion of the cell and aspiration pressure is characterized by the stiffness parameter, K(s) = 1.07 +/- 0.24 (SD) dyn/cm (n = 14). Values of K(s) do not correlate with the original cell length, which ranges from 29 to 74 microm. Theoretical analysis based on elastic shell theory applied to the experimental data yields an estimate of the effective elastic shear modulus, mu = 15.4 +/- 3.3 dyn/cm. These data were obtained at subcritical aspiration pressures, typically less than 10 cm H2O. After reaching a critical (vesiculation) pressure, the cytoplasmic membrane appeared to separate from the underlying structures, a vesicle with a length of 10-20 microm was formed, and the cytoplasmic membrane resealed. This vesiculation process was repeated until a cell-specific limit was reached and no more vesicles were formed. Over 20 vesicles were formed from the longest cells in the experiment.     

Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy

Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.     

Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.     

Cloned mice derived from somatic cell nuclei

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum.     

Vision-based force measurement using neural networks for biological cell microinjection

This paper presents a vision-based force measurement method using an artificial neural network model. The proposed model is used for measuring the applied load to a spherical biological cell during micromanipulation process. The devised vision-based method is most useful when force measurement capability is required, but it is very challenging or even infeasible to use a force sensor. Artificial neural networks in conjunction with image processing techniques have been used to estimate the applied load to a cell. A bio-micromanipulation system capable of force measurement has also been established in order to collect the training data required for the proposed neural network model. The geometric characterization of zebrafish embryos membranes has been performed during the penetration of the micropipette prior to piercing. The geometric features are extracted from images using image processing techniques. These features have been used to describe the shape and quantify the deformation of the cell at different indentation depths. The neural network is trained by taking the visual data as the input and the measured corresponding force as the output. Once the neural network is trained with sufficient number of data, it can be used as a precise sensor in bio-micromanipulation setups. However, the proposed neural network model is applicable for indentation of any other spherical elastic object. The results demonstrate the capability of the proposed method. The outcomes of this study could be useful for measuring force in biological cell micromanipulation processes such as injection of the mouse oocyte/embryo.     

Embryogenesis from microinjected single cells in a carrot cell suspension culture

A micrroinjection method was established for intact single cells with cell walls using a carrot suspension culture system in which selected single cells differentiate to embryos at high frequency. A solution of a fluorescent dye, Lucifer Yellow CH, was microinjected into those single cells, using an inverted microscope and a hydraulic micro-manipulator. In order to hold cells with cell walls and to overcome their turgor pressure, certain modification to conventional microinjection methods for protoplasts were necessary. The microinjected cells could divide and differentiate to embryos at a frequency of about 50%.     

An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells

The mechanical properties of endothelial cells were measured using the micropipette technique. The cells employed were collected from bovine aortic endothelium and cultured in our laboratory. Endothelial cells from confluent monolayers under no-flow conditions were detached from their substrate by trypsin or by a mechanical method and suspended in modified Dulbecco medium (MDM). In the micropipette technique, a part of the cell is aspirated into the tip of the micropipette under a microscope, and the deformation measured from a photograph. In this study, the data obtained were analyzed using a model where the cytoskeletal elements, which are considered to be the primary stress bearing components, are assumed to reside in a submembranous, cortical layer. Detached cells were found to have almost homogeneous mechanical properties based on measurements from different regions of the surface of a single cell. However, a hysteresis loop was observed in the relation between pressure and cell deformation during the loading and unloading processes. The calculated elastic shear moduli obtained for the trypsin-detached cells were as much as 10-20 times larger than those of a red blood cell. Mechanically-detached cells had moduli approximately twice that of the trypsin detached cells. Passage time, i.e., cell culture age, had no influence on the mechanical properties of the trypsin-detached cells, but did have an effect on the mechanically-detached cells, with both the younger and older cells being somewhat stiffer.     

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (相似文献   

A power-law rheology-based finite element model for single cell deformation

Physical forces can elicit complex time- and space-dependent deformations in living cells. These deformations at the subcellular level are difficult to measure but can be estimated using computational approaches such as finite element (FE) simulation. Existing FE models predominantly treat cells as spring-dashpot viscoelastic materials, while broad experimental data are now lending support to the power-law rheology (PLR) model. Here, we developed a large deformation FE model that incorporated PLR and experimentally verified this model by performing micropipette aspiration on fibroblasts under various mechanical loadings. With a single set of rheological properties, this model recapitulated the diverse micropipette aspiration data obtained using three protocols and with a range of micropipette sizes. More intriguingly, our analysis revealed that decreased pipette size leads to increased pressure gradient, potentially explaining our previous counterintuitive finding that decreased pipette size leads to increased incidence of cell blebbing and injury. Taken together, our work leads to more accurate rheological interpretation of micropipette aspiration experiments than previous models and suggests pressure gradient as a potential determinant of cell injury.     

Characterizing Cell Adhesion by Using Micropipette Aspiration

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.     

Conifer somatic embryogenesis for studies of plant cell biology

Summary Embryogenic cultures have been produced for a wide range of conifers and current methods developed for spruce permit the maturation of high quality embryos that can be desiccated and then germinated to form plantlets. Embryogenic suspensions consisting of immature embryos are an excellent source of regenerable protoplasts. This review considers examples of applications of embryogenic suspension cultures for basic studies in three areas of plant cell biology. a) Immunofluorescence studies of microtubules in mitotic spruce cells reveal focused spindle poles at prophase and anphase, suggesting the presence of microtubule organizing centers (MTOCs). Antibodies known to recognize animal MTOCs do not stain the polar regions but do stain developing kinetochores. b) Embryo-derived protoplasts regenerate directly to somatic embryos. Fluorescence studies of the cytoskeleton in freshly derived protoplasts reveal random cortical microtubules and a fine network of actin filaments. During culture, protoplasts change shape and develop transverse cortical microtubule arrays. Embryonal cells of newly formed embryos possess distinctive arrays of cortical microtubules and networks of fine actin filaments while suspensor cells are characterized by transverse cortical microtubules and longitudinal actin cables. c) Transmission electron microscope studies of endocytosis in spruce protoplasts reveal an endocytotic pathway similar to that described previously for soybean. Uptake results are confirmed using high pressure freeze fixation instead of conventional chemical fixation. Presented in the Session-in-Depth Morphogenesis: Plant Cell and Tissue Differentiation at the 1994 Congress on Cell and Tissue Culture, Research Triangle Park, NC, June 4–7, 1994.     

Stress-free state of the red blood cell membrane and the deformation of its skeleton

The response of a red blood cell (RBC) to deformation depends on its membrane, a composite of a lipid bilayer and a skeleton, which is a closed, twodimensional network of spectrin tetramers as its bonds. The deformation of the skeleton and its lateral redistribution are studied in terms of the RBC resting state for a fixed geometry of the RBC, partially aspirated into a micropipette. The geometry of the RBC skeleton in its initial state is taken to be either two concentric circles, a references biconcave shape or a sphere. It is assumed that in its initial state the skeleton is distributed laterally in a homogeneous manner with its bonds either unstressed, presenting its stress-free state, or prestressed. The lateral distribution was calculated using a variational calculation. It was assumed that the spectrin tetramer bonds exhibit a linear elasticity. The results showed a significant effect of the initial skeleton geometry on its lateral distribution in the deformed state. The proposed model is used to analyze the measurements of skeleton extension ratios by the method of applying two modes of RBC micropipette aspiration.     

Expression of Kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with various electric properties: patch clamp combined with single cell RT-PCR study

This study is to explore a new method of investigating molecular basis for electrophysiological properties of early fetal cardiomyocytes. Single embryonic cardiomyocytes of mouse early developmental heart (E10.5) were obtained by a collagenase B digestion approach. After recording spontaneous action potential using whole cell patch clamp technique, the single cell was picked by a glass micropipette, followed by a standard RT-PCR to explore the expression levels of several ion channel genes. Three phenotypes of cardiomyocytes were demonstrated with distinct properties: ventricular-like, atrial-like, and pacemaker-like action potentials. Ventricular-like and atrial-like cells were characterized with much negative maximum diastolic potential (MDP) and a higher V(max) (maximum velocity of depolarization) compared to pacemaker-like cells. MDP of ventricular-like cells was the most negative. In parallel, stronger expression of SCN5a, SCN1b and Kir2.1 were observed in ventricular-like and atrial-like cells compared to that of pacemaker-like cells, where Kir2.1 in ventricular-like cells was the most abundant. Cardiomyocytes with distinct electrophysiological properties had distinct gene expression pattern. Single cell RT-PCR combined with patch clamp technique could serve as a precise detector to analyze the molecular basis of the special electrophysiological characteristics of cardiomyocytes.     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62.6%和86.0%。用核染料Hoechst 33342 对卵母细胞的去核效率进行验证,去核成功率达到73.3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback

We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed against the corresponding protein product to allow the detection of its distribution using standard immunochemical techniques. We have used this approach to compare the spatial and temporal distribution patterns of the RNA and protein products of the segmentation gene hunchback (hb) during the early stages of embryogenesis. This comparison revealed translational control of the maternally derived hb mRNA, which was difficult to detect by conventional techniques. The non-radioactive in situ hybridization method is as sensitive as conventional methods, but is faster and easier to perform. This may make it a useful tool for a variety of other systems.     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62·6%和86·0%。用核染料Hoechst33342对卵母细胞的去核效率进行验证,去核成功率达到73·3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

Alteration of red cell membrane viscoelasticity by heat treatment: effect on cell deformability and suspension viscosity

The membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc) were measured for normal, control human red blood cells (RBC) and for RBC heat treated (HT) at 48 degrees C. Three separate methods for the measurement of mu were compared (two used a micropipette and one employed a flow channel), and the membrane viscosity (n) was calculated from the relation n = mu. tc. The deformability of HT and control cells was evaluated using micropipette techniques, and the bulk viscosity of RBC suspensions at 40% hematocrit was measured. The shear elastic modulus, or "membrane rigidity", was more than doubled by heat treatment, although both the absolute value for mu and the estimate of the increase induced by heat treatment varied depending on the method of measurement. Heat treatment caused smaller increases in membrane viscosity and in membrane bending resistance, and only minimal changes in cell geometry. The deformability of HT cells was reduced: 1) the pressure required for cell entry (Pe) into 3 micrometers pipettes was increased, on average, by 170%; 2) at an aspiration pressure (Pa) exceeding Pe, longer times were required for cell entry into the same pipettes. However, when Pa was scaled relative to the mean entry pressure for a given sample (i.e, Pa/Pe), entry times were similar for control and HT cells. Bulk viscosity of HT RBC suspensions was elevated by approximately 12% on average (shear rates 75 to 1500 inverse seconds). These findings suggest that alteration of RBC membrane mechanical properties, similar to those induced by heat treatment, would most affect the in vivo circulation in regions where vessel dimensions are smaller than cellular diameters.