Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane.

Wen, Tao; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

doi:10.1073/pnas.84.12.4180

Cited by 133 publications

(86 citation statements)

References 28 publications

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“…As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12,14,15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells [16][17][18][19][20][21][22], A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19,20], However, it is important to relate these physical effects to the biological response of the cells.…”

Section: Introductionmentioning

confidence: 99%

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Hellman

Rau

Yoon

et al. 2008

Journal of Biophotonics

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Cell lysis and molecular delivery in confluent monolayers of PtK 2 cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3kDa) FITC-conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time-resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam-induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ w, max > 190 ± 20 kPa are immediately lysed while cells exposed to τ w, max > 18 ± 2kPa are necrotic and subsequently detach. Cells exposed to τ w, max in the range 8-18 kPa are viable and successfully optoporated with 3kDa Dextran molecules. Cells exposed to τ w, max < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam-induced shear stresses and subsequent cellular outcome.

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Section: Introductionmentioning

confidence: 99%

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Hellman

Rau

Yoon

et al. 2008

Journal of Biophotonics

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“…The laser microbeam system was similar to that described previously (42). Laser transection of MTs was performed on cells in Rose culture chambers using the fourth harmonic 266-nm wavelength from a short-pulsed Nd/YAG laser (model YG 481A; Quantel Corp., Tempe, AZ).…”

Section: Laser Transection Of Mtsmentioning

confidence: 99%

Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

Wen

Walter

Berns

1988

The Journal of Cell Biology

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Abstract.To study the possible mechanism of microtubule turnover in interphase cells, we have used the 266-nm wavelength of a short-pulsed Nd/YAG laser to transect microtubules in situ in PtK2 cells at predefined regions. The regrowth and shrinkage of the transected microtubules have been examined by staining the treated cells with antitubulin mAb at various time points after laser irradiation. The results demonstrate that microtubules grow back into the transected zones individually; neither simultaneous growth nor shrinkage of all microtubules has been observed. The half-time of replacement of laser-dissociated microtubules is observed to be '~10 min. On the other hand, exposure of the core of the microtubule, which is expected to consist almost completely of GDP-tubulin, by transecting the internal regions of the microtubule does not render the remaining polymer catastrophically disassembled, and most transected microtubules with free minus ends do not quickly disappear. Taken together, these results suggest that most microtubules in cultured interphase cells exhibit some properties of dynamic instability (individual regrowth or shrinkage); however, other factors in addition to the hydrolysis of GTP-tubulin need to be involved in modulating the dynamics and the stability of these cytoplasmic microtubules.M ICROTUBULES (MTs) t are one of the three major fibrillar systems of the cytoskeleton and play an important role in cell movement, determination of cell shape, organization of the internal architecture of the cell, and segregation of chromosomes in mitosis (10, 34). For a better understanding of these fundamental cellular processes it is essential to understand the mechanism of assembly of the MT polymer.MTs were initially postulated to be polymers in a simple equilibrium with the free tubulin subunits (18, 30). Subsequently, evidence indicated that this view was overly simple. Experimental as well as theoretical explorations of the role of nucleotide hydrolysis in assembly led to the treadmilling model which proposes that, at steady state, there may be a net tubulin addition at one end of the polymer and a net loss from the other end, resulting in a unidirectional flux of tubulin through MTs (9, 25; reviewed in reference 26). More recently, based on observations of individual MT behavior under conditions in which the free monomer concentration is equal to or below the steady-state concentration and on modeling of dynamics of theoretical MTs using hypothetical values for the rate constants, an alternative "dynamic instability" model was proposed. This model asserts that over a wide tubulin concentration range, a slow growing phase and rapid shrinking phase may coexist in a population of MTs;1. Abbreviation used in this paper: MT, microtubule. growing MTs are postulated to have tubulin subunits with bound GTP at the polymer ends, while the terminal subunits in shrinking MTs are thought to have bound GDP. The two phases interconvert stochastically and infrequently (15,16,23,27,28).At present, it appears tha...

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“…This technology platform has been used to implement numerous forms of laser-mediated cell manipulation in an automated high-throughput fashion, thereby overcoming many of the practical implementation and throughput issues surrounding previous reports of such techniques (12,(21)(22)(23). This report details the use of LEAP for in situ cell imaging and laser-mediated cell purification, demonstrating compelling advantages for several applications that are not well-served by existing approaches.…”

Section: Discussionmentioning

confidence: 99%

“…Building on the knowledge from medical laser uses and robust high-throughput semiconductor manufacturing tools, a novel laser-mediated cell manipulation technology has been developed. The resulting laser-enabled analysis and processing (LEAP) technology can implement cell imaging and multiple forms of laser-mediated cell manipulations including cell purification, optoinjection (i.e., laser-mediated cell transfection) (18,19), and chromophore-assisted laser-inactivation (e.g., direct knockout of protein function) (20) in an automated and highthroughput manner, thereby overcoming certain limitations of previous approaches to these methods (12,(21)(22)(23).…”

mentioning

confidence: 99%

High‐throughput laser‐mediated in situ cell purification with high purity and yield

et al. 2004

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Background: Technologies for purification of living cells have significantly advanced basic and applied research in many settings. Nevertheless, certain challenges remain, including the robust and efficient purification (e.g., high purity, yield, and sterility) of adherent and/or fragile cells and small cell samples, efficient cell cloning, and safe purification of biohazardous cells. In addition, existing purification methods are generally open loop and exhibit an inverse relation between cell purity and yield. Methods: An automated closed-loop (i.e., employing feedback control) cell purification technology was developed by building upon medical laser applications and laser-based semiconductor manufacturing equipment. Laser-enabled analysis and processing has combined highthroughput in situ cell imaging with laser-mediated cell manipulation via large field-of-view optics and galvanometer steering. Laser parameters were determined for cell purification using three mechanisms (photothermal, photochemical, and photomechanical), followed by demonstration of system performance and utility. Results: Photothermal purification required approximately 10 8 W/cm 2 at 523 nm in the presence of Allura Red, resulting in immediate protein coagulation and cell necrosis. Photochemical purification required approximately 10 9 W/cm 2 at 355 nm, resulting in apoptosis induction over 4 to 24 h. Photomechanical purification

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Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane.

Cited by 133 publications

References 28 publications

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Laser-transected microtubules exhibit individuality of regrowth, however most free new ends of the microtubules are stable.

High‐throughput laser‐mediated in situ cell purification with high purity and yield

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