Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3270-3271
CORRESPONDENCE
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To the Editor: |
Gentle microinjection for myeloid cells using SLAM
The ability to microinject small, nonadherent blood
cells with macromolecules promises to open a number of new avenues of research and of potential therapy. The recent paper by Davis et al1 demonstrates both the need for the development of such techniques and the difficulty in so doing. Davis et al used "glass needles" (or micropipettes) to inject material into small cells by
stabbing the cells and applying a pulse of pressure to eject the
material (nucleic acid) from the micropipette. Glass-based microinjection is not new, and although the authors referred to papers
in the 1970s,2,3 the technique is much older, with applications dating back at least to the 1920s.4 Not
surprisingly for technology nearly 80 years old, there are limitations
with the glass "stab injection" method. There are two fundamental
problems that have limited the usefulness of this approach to blood
cell research.
The first problem is the need for the cell to be firmly immobilized to
withstand the stabbing action of the micropipette. Davis et
al1 elegantly overcame this problem by temporarily immobilizing the cells while injection was occurring. Although it may
be questioned whether a procedure for firmly adhering the cells which
resulted in cell shape change and micropodia formation was neutral to
the cell physiology, it at least permitted the microinjection to be
performed. The second problem is the damage caused to the
cell by the insertion of the glass micropipette through the plasma
membrane and into the cytosol. There are two types of cellular damage
that occur during microinjection, one associated with the breaking into
the cell (mechanical damage to the cell surface) and the other
associated with the presence of the glass in the cytosol (protein
denaturation and coagulation on the glass). Although the plasma
membrane may readily "seal" after microinjection, the effect of
the glass on the cytosol can be less tolerable. While limited cytosolic
damage can be tolerated by cells, the percentage of damage inflicted by
this "glass-stab injection" increases as the size of the cell
decreases. Thus a large cell of diameter 100 µm may tolerate the
insult of microinjection if only 0.01% of its volume experiences the
damage. But with the same injection process on a cell which is 1/10th
this size (ie, with a spherical diameter of 10 µm, having a volume
1/1000th that of the 100-µm cell), the damage resulting from
microinjecting the smaller cell will be 1000 times greater (ie, 10% of
its volume will experience the effect of microinjection). Even with a
micropipette of 0.2-µm tip diameter (as opposed to a "standard"
0.5 µm), the improvement in reducing glass-volume-related damage is
only a factor of 2.5. It can thus be appreciated why Davis et
al1 must be congratulated on achieving such "good"
survival rates as 50%, with only 10% to 20% of cells showing
immediate signs of damage from microinjection.
But there is now an alternative procedure for microinjecting nucleic
acid, proteins, and other membrane impermeant molecules into small
cells, a method that overcomes the two major problems with traditional
microinjection. The new method does not require the cells to be firmly
adhered to a substrate, and the glass micropipette does not penetrate
the cell. This procedure has been used with a number of cell types,
including blood cells, and has been demonstrated in detail with human
neutrophils.5 The new approach is based on a modification
of the standard microinjection procedure and is called SLAM (simple
lipid-assisted microinjection). In essence, the tip of the glass
micropipette is coated with a lipid bilayer to produce a "soft"
non-cell-penetrating tip.5,6 On contact between the lipid
bilayers at the tip of the micropipette and the cell membrane, fusion
between the lipid bilayers occurs, and an aqueous channel is formed
between the inside of the micropipette and the cytosol. Material inside
the micropipette therefore gains access to the inside of the blood cell
either by diffusion or by gentle ejection with low
pressures.5 Removal of the SLAM pipette permits the lipid
bilayers to reform. There is no need for the cell to be firmly attached
to a substrate because there is no sudden movement of the micropipette.
There is no mechanical damage to the plasma membrane, and because the
glass of the SLAM-pipette does not enter the cell cytosol, no
glass-induced damage occurs inside the cell. Not only does this
procedure result in extremely high cell survival (at least 80% to
90%), but it can be used on cells that have hitherto been impossible
to microinject.
A comparison between the stab injection and the SLAM injection methods
was made recently by Carmichael7 by considering the methods
from the cell's point of view (if it has one). The stab injection when
scaled up to human size is likened to a 40-mph advancing basketball
that must be stopped precisely to achieve its goal. But with SLAM
injection a gentle touch (to ensure contact between the two lipid
bilayers) is all that is required. The cells do not need to adhere to a
surface, although a loose adherence is useful to avoid
the cell being moved by currents in the medium as the micropipette
advances. The health of SLAM-injected neutrophils is demonstrable by
their ability to maintain normal cytosolic-free Ca2+
concentration,8 and the lack of damage can be shown by
choosing to microinject neutrophils with forming cytosolic ruffling
extensions that are very responsive to damage and that retract and
round up during attempts at standard stab injection. The Figure
shows such a SLAM injection in which the
neutrophil maintains its shape and the ruffling of the membrane
persists.
View larger version (114K):
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A sequence of images illustrating the gentle nature of
SLAM-injection.
The uppermost 2 images show a human neutrophil that is loosely adhered
and ruffling at the left-hand edge and at the beginning of the
microinjection process. The SLAM injector contains lucifer yellow as a
fluorescent (non-protein-binding) marker. The middle panel shows 3 fluorescent images (left) at contact between the SLAM-injector tip (in
focus and brightly glowing) and then 8 seconds (middle) and 14 seconds
(right) later. The transfer of fluorescent material into the cell can
be seen. There is no indication of cell damage or response to the SLAM
injection as evidenced by cell-shape change or recoiling of the ruffled
membrane. The lowest panel shows the phase-contrast images at the end
of SLAM injection, when a high level of uniform intracellular
fluorescence is seen. (See Hallett and Laffafian9 for a
movie of other SLAM injections.)
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An additional bonus resulting from the SLAM technique is the ability to
modify and modulate the composition of the plasma membrane. If
bioactive lipids are included in the slam coating, they are transferred
to the cell membrane. Also, it may be possible to incorporate
membrane-associated proteins into the SLAM bilayer for its transfer to
the cell membrane (with its orientation defined by which side of the
bilayer in which is incorporated). Thus the development of new
techniques for microinjection (and manipulation of plasma membrane) of
blood cells promises to permit a new strategy for the investigation of
blood cell physiology.
Iraj Laffafian
Maurice B. Hallett
Molecular Signalling Group
University Department of
Surgery
University of Wales College of Medicine
Heath Park,
United Kingdom
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References |
1.
Davis BR, Yannariello J, Prokopishyn NL, et al.
Class needle-mediated micro-injection of macromolecules and transgenes into primary human blood stem/progenitor cells.
Blood.
2000;95:437-444[Abstract/Free Full Text].
2.
Diacumakos EG, Holland S, Pecora P.
A microsurgical methodology for human cells in vitro: evolution and applications.
Proc Natl Acad Sci U S A.
1970;65:911-918[Abstract/Free Full Text].
3.
Grassmann A.
Microsurgical cell nucleus transplantation in mammalian cells.
Exp Cell Res.
1970;60:373-382[Medline]
[Order article via Infotrieve].
4.
Chambers R, Pollack H, Hillier S.
The protoplasmic pH of living cells.
Proc Soc Exp Biol Med.
1927;24:760-771.
5.
Laffafian I, Hallett MB.
Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.
Biophys J.
1998;75:2558-2563[Abstract/Free Full Text].
6.
Peters RN, Sikorski R.
The gentle slam.
Science.
1998;282:2213-2214.
7.
Carmichael SW.
How to SLAM a cell.
Microscopy Today.
1999;99:3.
8.
Hallett MB, Hodges R, Cadman M, et al.
Techniques for measuring and manipulating free Ca2+ in the cytosol and organelles of neutrophils.
J Immunol Methods.
1999;232:77-88[Medline]
[Order article via Infotrieve].
9. Hallett MB and Laffafian. SLAM cell engineering. Available at:
http://www.uwcm.ac.uk/uwcm/sr/slam.html. Accessed January
25, 2000.
