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Biomedical subjects

G P Li

Publications and source records attributed to G P Li.

At least 19 recordsLinked to original sources
PubMed2007

Micropallet arrays for the separation of single, adherent cells.

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.

Animals↗
PubMed2006

Micropatterning of living cells on a heterogeneously wetted surface.

We report a simple approach to fabricate heterogeneously wetted surfaces which can be used to pattern living cells or biomolecules. An array of pedestals composed of SU-8 was fabricated on a glass surface which was then derivatized with a hydrophobic silane. Upon addition of aqueous solutions to the array, air was trapped within the hydrophobic cavities between the pedestals. The trapped air formed a "virtual wall" blocking access to these cavities. Cells cultured on the array were forced to grow only on the tops of the pedestals, i.e., the surfaces wetted by aqueous media. The virtual walls were stable during manipulation of the array and over long time periods (months).

Air↗
PubMed2006

Simple photografting method to chemically modify and micropattern the surface of SU-8 photoresist.

SU-8 has gained widespread acceptance as a negative photoresist. It is also finding increasing use as a structural material in microanalytical devices. Consequently, methods to tailor the surface properties of SU-8 as well as to micropattern coatings on the surface of SU-8 are needed. The SU-8 photoresist consists of EPON SU-8 resin mixed with the photoacid generator triarylsulfonium hexafluoroantimonate. This photoacid generator can also serve as a photoinitiator generating free radicals when illuminated with UV light. Under the appropriate conditions, sufficient triarylsulfonium hexafluoroantimonate remains within cured SU-8 to act as a source of free radicals and initiate UV-mediated grafting of polymers onto the surface of the SU-8. UV-mediated grafting was used to coat SU-8 surfaces with poly(acrylic acid) and other water-soluble monomers. The SU-8 surface was chemically micropatterned by placing a mask between the UV light and SU-8. The X-Y spatial resolution of micropatterned poly(acrylic acid) on the SU-8 surface was 2 mum. Three applications of these chemically modified SU-8 surfaces were demonstrated. In the first, poly(ethylene glycol) was used to protect the SU-8 from interactions with proteins, yielding a surface resistant to biofouling. In the second demonstration, the SU-8 surface was micropatterned with a cell-resistant layer to guide cellular attachment and growth. In the final application, SU-8 micropallets were encoded with polymer lines. The bar codes were read by either absorbance or fluorescence measurements. Thus, UV-mediated graft polymerization is an efficient and effective method to micropattern coatings onto the surface of SU-8.

Animals↗
PubMed2006

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos.

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

Animals↗
PubMed2005

Covalent micropatterning of poly(dimethylsiloxane) by photografting through a mask.

A new photografting method to micropattern a covalent surface modification on poly(dimethylsiloxane) (PDMS) provides advantages in simplicity and efficiency. To accomplish the entire process on the benchtop, the PDMS was initially treated with benzophenone dissolved in a water/acetone mixture. This process permitted limited diffusion of the photoinitiator into the PDMS surface. Polymerization of acrylic acid was initiated by exposure of the benzophenone-implanted PDMS to UV radiation through a photomask with a thin aqueous layer of acrylic acid sandwiched between the PDMS and photomask. This procedure resulted in patterned poly(acrylic acid) (PAA) on the PDMS surface. In the modified regions, PAA and PDMS formed an interpenetrating polymer network extending 50 microm into the PDMS with an X-Y spatial resolution of 5 microm. The carboxyl groups of the PAA graft could be derivatized to covalently bond other molecules to the patterned PAA. Two bioanalytical applications of this micropatterned surface were demonstrated: (1) a guide for cell attachment and growth and (2) a substrate for immunoassays. 3T3 cells were shown to selectively localize to modified surface regions where they could be cultured for up to 7 days. Additionally, the micropatterned surface was used to immobilize either protein A or antibody for heterogeneous immunoassays.

Acrylic Resins↗
PubMed2005

Characteristic dysfunction of stunned myocardium induced by 2,3-butanedione monoxime without ischaemia.

1. In the present study, we tested the hypothesis that, even in the absence of prior ischaemia, 2,3-butanedione monoxime (BDM), an inhibitor of contraction at the actin-myosin level, could produce the postischaemic dysfunction characteristic of stunned myocardium. 2,3-Butanedione monoxime was injected directly into the left anterior descending coronary artery (LAD) before and again after myocardial stunning produced by 15 min occlusion of the LAD followed by 30 min reperfusion. 2. Regional myocardial force, segment shortening and regional work were measured in both the LAD-perfused area and the area perfused by the circumflex coronary artery, which served as a control area. Regional dysfunction produced by BDM injection or ischaemia-reperfusion was assessed quantitatively by five parameters: end-diastolic length (EDL), shortening onset delay (delay), systolic bulge (bulge), end-shortening time delay (EST) and tail work ratio (TWR). 3. It was found that injection of BDM into the LAD caused dyskinesis similar to that caused by occlusion-reperfusion. Both displayed elevated EDL and marked increases in delay, bulge, EST and TWR; these parameters were significantly higher in the dyskinesis caused by BDM injection. Despite dysfunctional fibre shortening, intracoronary BDM injection did not reduce regional force. 4. Thus, BDM can elicit changes similar to those characteristic of postischaemic dysfunction. Because contractility was not impaired, dysfunction was apparently caused by disrupting the association between contractile force and muscle motion.

Acetylcholine↗
PubMed2004

Tailoring the surface properties of poly(dimethylsiloxane) microfluidic devices.

Poly(dimethylsiloxane) (PDMS) is an attractive material for microelectrophoretic applications because of its ease of fabrication, low cost, and optical transparency. However, its use remains limited compared to that of glass. A major reason is the difficulty of tailoring the surface properties of PDMS. We demonstrate UV grafting of co-mixed monomers to customize the surface properties of PDMS microfluidic channels in a simple one-step process. By co-mixing a neutral monomer with a charged monomer in different ratios, properties between those of the neutral monomer and those of the charged monomer could be selected. Mixtures of four different neutral monomers and two different charged monomers were grafted onto PDMS surfaces. Functional microchannels were fabricated from PDMS halves grafted with each of the different mixtures. By varying the concentration of the charged monomer, microchannels with electrophoretic mobilities between +4 x 10(-4) cm2/(V s) and -2 x 10(-4) cm2/(V s) were attainable. In addition, both the contact angle of the coated surfaces and the electrophoretic mobility of the coated microchannels were stable over time and upon exposure to air. By carefully selecting mixtures ofmonomers with the appropriate properties, it may be possible to tailor the surface of PDMS for a large number of different applications.

Dimethylpolysiloxanes↗
PubMed2004

Surface-directed, graft polymerization within microfluidic channels.

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.

Journal Article↗
PubMed2003

Fast electrical lysis of cells for capillary electrophoresis.

In the past decade, capillary electrophoresis has demonstrated increasing utility for the quantitative analysis of single cells. New applications for the analysis of dynamic cellular properties demand sampling methods with sufficient temporal resolution to accurately measure these processes. In particular, intracellular signaling pathways involving many enzymes can be modulated on subsecond time scales. We have developed a technique to rapidly lyse an adherent mammalian cell using a single electrical pulse followed by efficient loading of the cellular contents into a capillary. Microfabricated electrodes were designed to create a maximum voltage drop across the flattened cell's plasma membrane at a minimum interelectrode voltage. The influence of the interelectrode distance, pulse duration, and pulse strength on the rate of cell lysis was determined. The ability to rapidly lyse a cell and collect and separate the cellular contents was demonstrated by loading cells with Oregon Green and two isomers of carboxyfluorescein. All three fluorophores were detected with a separation efficiency comparable to that of standards. Parallel comparison of electrical lysis to that produced by a laser-based lysis system revealed that the sampling efficiencies of the two techniques were comparable. Rapid cell lysis by an electrical pulse may increase the application of capillary electrophoresis to the study of cellular dynamics requiring fast sampling times.

Animals↗
PubMed2003

Cross-linked coatings for electrophoretic separations in poly(dimethylsiloxane) microchannels.

We have developed a strategy using ultraviolet light to polymerize mixed monomer solutions onto the surface of a poly(dimethylsiloxane) (PDMS) microdevice. By including monomers with different chemical properties, electrophoretic separations were optimized for a test set of analytes. The properties of surfaces grafted with a single neutral monomer, a neutral and a negative monomer, or a neutral, negative, and cross-linking monomer were assessed. The highest quality separations were achieved in channels with cross-linked coatings. The separation efficiency for biologically relevant peptides (kinase substrates) on these surfaces was as high as 18 600 theoretical plates in a 2.5 cm channel. The test peptides were fluorescein-AEEEIYGEFEAKKKK, fluorescein-GRPRAATFAEG, fluorescein-GRPRAA(T-PO(3))FAEG, fluorescein-DLDVPIP GRFDRRVSVAAE, and fluorescein-DLDVPIPGRFDRRV(S-PO(3))VAAE. Separations between two different peptides occurred in as little as 400 ms after injection into the separation channel. The simultaneous separation of five kinase and phosphatase substrates was also demonstrated. By carefully selecting mixtures of monomers with the appropriate properties, it may be possible to tailor the surface of PDMS for a large number of different electrophoretic separations.

Amino Acid Sequence↗
PubMed2002

Surface modification of poly(dimethylsiloxane) microfluidic devices by ultraviolet polymer grafting.

Poly(dimethylsiloxane) (PDMS)-based microfluidic devices are increasing in popularity due to their ease of fabrication and low costs. Despite this, there is a tremendous need for strategies to rapidly and easily tailor the surface properties of these devices. We demonstrate a one-step procedure to covalently link polymers to the surface of PDMS microchannels by ultraviolet graft polymerization. Acrylic acid, acrylamide, dimethylacrylamide, 2-hydroxylethyl acrylate, and poly(ethylene glycol)monomethoxyl acrylate were grafted onto PDMS to yield hydrophilic surfaces. Water droplets possessed contact angles as low as 45 degrees on the grafted surfaces. Microchannels constructed from the grafted PDMS were readily filled with aqueous solutions in contrast to devices composed of native PDMS. The grafted surfaces also displayed a substantially reduced adsorption of two test peptides compared to that of oxidized PDMS. Microchannels with grafted surfaces exhibited electroosmotic mobilities intermediate to those displayed by native and oxidized PDMS. Unlike the electroosmotic mobility of oxidized PDMS, the electroosmotic mobility of the grafted surfaces remained stable upon exposure to air. The electrophoretic resolution of two test peptides in the grafted microchannels was considerably improved compared to that in microchannels composed of oxidized PDMS. By using the appropriate monomer, it should be possible to use UV grafting to impart a variety of surface properties to PDMS microfluidics devices.

Dimethylpolysiloxanes↗
PubMed2002

The mass transfer process and the growth rate of protein crystals.

In this paper, protein crystal growth is studied by a Mach-Zehnder interferometer and an image process system. The interference fringe images are recorded during the crystallization of tetragonal hen egg white lysozyme crystal. Concentration distributions of the protein solution are given from the interference fringe images recorded by the Mach-Zehnder interferometer with a real time servo system of a four-step phase shift. The mass transfer flux and the crystal growth rates are obtained from the concentration distribution. The results show that the observed rates are in accordance with those demonstrated by measurements of the experimental images; therefore the method for determining growth rate by the diffusion process is reasonable.

Crystallization↗
PubMed2001

Electroosmotic properties of microfluidic channels composed of poly(dimethylsiloxane).

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH. oxidized PDMS had a four-fold greater EO mobility (mu(eo)) compared to native PDMS. The greater mu(eo) was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced mu(eo) was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the mu(eo).

Dimethylpolysiloxanes↗
PubMed2001

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Cu,Zn superoxide dismutase from Peking duck.

The cDNA encoding Peking duck Cu,Zn superoxide dismutase (dSOD) was cloned and sequenced. The recombinant enzyme was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion technique. Trigonal crystals of dSOD were obtained at 278 K at low ionic strength and around neutral pH. These crystals belong to space group P3(2)21, with unit-cell parameters a = 124.4, c = 163.5 A, gamma = 120 degrees. The asymmetric unit contains four dimers (eight monomers of Cu,Zn dSOD) and has a 56% solvent content, with a V(M) of 2.8 A(3) Da(-1). On a Rigaku R-AXIS IIc image-plate area-detector system, the crystal diffracted to 2.9 A. Unusual supermolecular double-helix packing with 9(2)2 non-crystallographic symmetry in crystals has been observed in the initial structural analysis.

Amino Acid Sequence↗
PubMed2001

Maturation of the reconstructed oocytes by germinal vesicle transfer in rabbits and mice.

The present study was designed to evaluate the feasibility of germinal vesicle (GV) transfer in rabbits and mice. The GV oocytes were collected from ovaries and cultured in 20 microg/mL 3-isobutyl-1-methylxanthin (IBMX) in TCM199 medium, which caused oocytes to shrink, enlarging the perivitelline space to facilitate the GV removal and transfer. Pairs of GV-cytoplast complexes were fused with electric pulses, and the fused, reconstructed oocytes were cultured in TCM199 for 24 h. Results are as follows: 1) The exposure time of rabbit GV oocytes to IBMX medium affected the success of GV removal. For oocytes cultured for 2 and 3 h in IBMX medium, removed rates were 56% and 44, respectively, significantly higher (P < 0.05) than removal rates of GV oocytes cultured for 1 and 4 h (27% and 27%, respectively); 2) There was no significant difference (P > 0.1) in fusion and maturation rates of rabbit reconstructed oocytes collected at 72 and 84 h after initiation of FSH injection to donors; 3) eCG in the maturation media improved development of rabbit-to-rabbit GV transferred oocytes but had no positive effect on mouse-to-rabbit GV transferred oocytes; 4) When mouse GV-karyoplasts were injected into enucleated rabbit oocytes, fusion rates of GV-karyoplasts measuring 40- to 50-microm and 80- to 90-microm in diameters obtained were 84% and 93%, respectively. The rates were significantly higher (P < 0.05) than fusion rates after transferring GV-karyoplasts measuring 30- to 35-microm in diameter (63%). The maturation rate (89%) of reconstructed oocytes composed of 80- to 90-microm mouse GV-karyoplasts and rabbit GV-enucleated cytoplasts was higher than that seen for oocytes composed of 40- to 50-microm (77%, P<0.05) or 30- to 35-microm (59%, P<0.01) mouse karyoplasts. Thirty-five of the 63 (56%) mature mouse-to-rabbit reconstructed oocytes had the normal complement of 20 chromosomes.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Crystallization and preliminary X-ray analysis of a depressant insect toxin from the scorpion Buthus martensii Karsch.

Depressant insect toxins are a distinct group of scorpion neurotoxins for which no three-dimensional structures are yet available. A depressant insect toxin named BmK dITAP3 from the scorpion Buthus martensii Karsch (BmK) has been purified and crystallized. Single crystals of dITAP3 grew in the presence of the detergent CYMAL-6 using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. A set of diffraction data to 2.6 A resolution has been collected. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group R3, with unit-cell parameters a = b = 73.29, c = 68.90 A, alpha = beta = 90, gamma = 120 degrees. Assuming two molecules in the asymmetric unit, the estimated solvent content is 53.4%.

Animals↗
PubMed2001

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals.

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.

Animals↗