U.S. patent application number 15/171324 was filed with the patent office on 2016-09-29 for methods and devices for electroporation.
The applicant listed for this patent is JIAN CHEN. Invention is credited to JIAN CHEN.
Application Number | 20160281047 15/171324 |
Document ID | / |
Family ID | 47744254 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160281047 |
Kind Code |
A1 |
CHEN; JIAN |
September 29, 2016 |
METHODS AND DEVICES FOR ELECTROPORATION
Abstract
An apparatus for electroporation of biological cells is
provided. The apparatus includes a sample container having an
insulator chamber for holding the cells. The sample container has a
first electrode and a second electrode to provide electrical
connection for electroporation. The insulator chamber is configured
to contain at least one cell monolayer. The apparatus also includes
a pulse generator that can generate a predetermined pulse for
electroporation of the cells.
Inventors: |
CHEN; JIAN; (Blue Bell,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHEN; JIAN |
Blue Bell |
PA |
US |
|
|
Family ID: |
47744254 |
Appl. No.: |
15/171324 |
Filed: |
June 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13594730 |
Aug 24, 2012 |
9382510 |
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15171324 |
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61527357 |
Aug 25, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 13/00 20130101;
C12N 15/87 20130101; C12M 35/02 20130101 |
International
Class: |
C12M 1/42 20060101
C12M001/42; C12N 15/87 20060101 C12N015/87; C12N 13/00 20060101
C12N013/00 |
Claims
1-33. (canceled)
34. An apparatus for electroporation of biological cells,
comprising: a sample container having an insulator chamber for
holding cells, the sample container having a first electrode and a
second electrode providing electrical connection for
electroporation; a first conductive medium layer containing an
aqueous solution having solute ions; and a surface formed on the
first conductive medium layer, wherein: the surface is configured
to support at least one cell monolayer which is separated from the
first electrode and the second electrode, and the first conductive
medium layer directly conduct an electric pulse through the solute
ions to the at least one cell monolayer through substantially the
entire surface.
35. The apparatus of claim 34, wherein: at least one of the first
electrode and the second electrode is a movable electrode and
serves as a sealing cover confining a cell-containing sample within
the insulator chamber and between the first electrode and the
second electrode during the electrical pulse.
36. The apparatus of claim 34, wherein: the insulator chamber has a
groove on a first end of a wall of the insulator chamber for
receiving extra liquid.
37. The apparatus of claim 34, further comprising: a precast porous
matrix, wherein: the first conductive medium layer is cast in the
porous matrix.
38. The apparatus of claim 34, further comprising: a centrifuge
configured to assist formation of the at least one cell monolayer
by centrifugation.
39. The apparatus of claim 38, further including: a pulse generator
configured to deliver an electric pulse to the sample container for
electroporation during rotation.
40. The apparatus of claim 34, further comprising: a plurality of
artificial insulator particles to form the at least one cell
monolayer with the cells.
41. The apparatus of claim 39, wherein: the pulse generator has a
rechargeable battery for cordless function.
42. The apparatus of claim 38, wherein: the pulse generator for
generating the electrical pulse is integrated to the
centrifuge.
43. The apparatus of claim 37, wherein: the porous matrix is
selected from the group consisting of agar, agarose based gel,
silicone gel, polyacrylamide gel, collagen, gelatin gel, matrigel,
hyaluronic acid gel, alginate gel, polyethylene glycol gel, methyl
cellulose, modified cellulose based gel, acrylates gel, polyglycols
gel, propylene glycol gel, silicone, resins, glass fibers,
polymethacrylates, silicates, modified cellulose, polyvinyls,
polylysine, polyacrylic acid, polyethylene glycol, polyacrylamides
and co-polymers.
44. A process for electroporation of biological cells, comprising:
providing a sample container for electroporation, the sample
container having an insulator chamber for containing a cell sample,
a first electrode and a second electrode providing electrical
connection for electroporation, and a first conductive medium layer
for forming a surface which is separated from the first electrode
and the second electrode, arranging cells to form at least one cell
monolayer on the surface of the first conductive medium layer, the
at least one cell monolayer being separated from the first
electrode and the second electrode, treating the cells in the at
least one cell monolayer with a predetermined electrical pulse, the
predetermined electrical pulse being generated by a pulse
generator.
45. The process of claim 44, further comprising: sealing the cell
sample in the sample container by a tight closure.
46. The process of claim 44, wherein: the cells are arranged to the
at least one cell monolayer by natural gravity.
47. The process of claim 44, wherein: the cells are arranged to the
at least one cell monolayer by centrifugation.
48. The process of claim 47, wherein: the cells are treated with
the predetermined electrical pulse during the centrifugation.
49. A process for electroporation of biological cells, comprising:
providing a sample container for electroporation, the sample
container having an insulator chamber for forming the body of the
container to hold cells, a first electrode and a second electrode
providing electrical connection for electroporation, loading a
sample containing biological cells to the sample container, the
sample forming a convex surface after loading into the sample
container, forming a tight seal of the sample by the insulator
chamber, the first electrode and the second electrode, an extra
volume of the sample under the convex surface being pushed out by
the sealing action, treating the biological cells in the sample
container confined between the insulator chamber and the two
electrodes with a predetermined electrical pulse.
50. The process of claim 49, wherein: the first electrode is in a
cover configured to removably cover the insulator chamber, and the
insulator chamber is configured to contain a groove on a top of one
side of the insulator chamber, the cover being shaped to fit with
the groove to seal the insulator chamber, the first electrode
covering an inner rim of the groove on the side of the insulator
chamber, the extra sample pushed out by sealing of the sample
container is contained in the groove.
51. The process of claim 49, wherein: the first electrode and the
second electrode are fixed in the insulator chamber, a groove is
positioned on one side of the insulator chamber and on the
electrodes, and an insulator cover is configured to removably cover
the insulator chamber, the extra sample pushed out by sealing of
the sample container is contained in the groove.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority of U.S. Provisional Patent
Application No. 61/527,357, filed on Aug. 25, 2011, the entire
contents of which are incorporated by reference herein.
FIELD OF INVENTION
[0002] This invention relates generally to methods and devices for
electrical stimulation of cells and, more particularly, to methods
and devices for electroporation of cells.
BACKGROUND
[0003] Electroporation is a widely-used method for permeabilization
of cell membranes by temporary generation of membrane pores with
electrical stimulation. The applications of electroporation include
the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies,
drugs or other substances to a variety of cells such as mammalian
cells, plant cells, yeasts, other eukaryotic cells, bacteria, other
microorganisms, and cells from human patients. Electrical
stimulation may also be used for cell fusion in the production of
hybridomas or other fused cells. Electrical cell fusion may be
regarded as a special form of electroporation.
[0004] During a typical electroporation, cells are suspended in a
buffer or medium that is favorable for cell survival. For bacterial
cells electroporation, low conductance medium, such as water, is
often used to reduce the heat production by transient high current.
The cell suspension is then placed in a rectangular cuvette
embedded with two flat electrodes for an electrical discharge. For
example, Bio-Rad (Hercules, Calif.) makes Gene Pulser line of
products to electroporate cells in cuvettes. Traditionally,
electroporation requires high field strength.
[0005] The electroporation process is usually toxic to the cells.
First, when the electric field strength is too high, the cell
membranes may be irreversibly damaged. Secondly, while electrically
induced membrane pores allow a target substance to enter the cells,
the pores may also allow outflow of cellular contents and inflow of
other unintended substances which could negatively affect cell
viability. Thirdly, the heat generated by the electric current may
harm the cells. Lastly, electrochemically generated toxic agents
such as free radicals, gas and metal ions near the electrodes are
harmful to the cells.
[0006] Variation of cellular properties, i.e., heterogeneity of
cells during electroporation remains the biggest hurdle for
achieving high-efficiency electroporations with low cellular
toxicities. One known factor contributing to the heterogeneity is
cell size. Larger cells tend to be easier to be electroporated. For
a mixture of cells with different sizes, when larger cells are
efficiently electroporated under certain voltage, the voltage is
often not sufficient to electroporate smaller cells efficiently. At
a field strength that smaller cells are efficiently electroporated,
larger cells are usually irreversibly damaged because the voltage
is usually too high for the larger cells to survive. Other factors,
such as different cell membrane composition or cell maturity, may
also contribute to the heterogeneity of cells.
[0007] Despite of numerous attempts to improve the efficiency of
cell electroporations, the critical problem of cell heterogeneity
remains unsolved. The efficiency, cell survivability and cost
effectiveness of electroporation methods can be further improved.
The disclosed devices and methods are directed at solving one or
more problems set forth above and other problems.
BRIEF SUMMARY OF THE DISCLOSURE
[0008] One aspect of the present disclosure provides an apparatus
for electroporation of biological cells. The apparatus includes a
sample container having an insulator chamber for holding the cells.
The sample container has a first electrode and a second electrode
to provide electrical connection for electroporation. The insulator
chamber is configured to contain at least one cell monolayer. The
apparatus also includes a pulse generator that can generate a
predetermined pulse for electroporation of the cells.
[0009] Another aspect of the present disclosure provides an
apparatus for electroporation of biological cells. The apparatus
includes a sample container for holding a sample of biological
cells for electroporation. The container includes an insulator
chamber that forms the body of the container to hold the cells. The
insulator chamber has a plurality of sides. The container also
includes a first electrode and second electrode to receive an
electrical pulse from an electrical pulse generator to
electroporate the cells. The insulator chamber and the electrodes
are able to seal the sample of biological cells within the sample
container.
[0010] Another aspect of the present disclosure provides a process
for electroporation of biological cells. The process includes the
following steps. The cells are arranged to form at least one cell
monolayer in an insulator chamber of a sample container. The sample
container has a first electrode and a second electrode to provide
electrical connection for electroporation. The cells in the cell
monolayer are treated with a predetermined electrical pulse, which
is generated by a pulse generator.
[0011] Other aspects of the present disclosure can be understood by
those skilled in the art in light of the description, the claims,
and the drawings of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A illustrates an exemplary apparatus for
electroporation of biological cells consistent with the disclosed
embodiments;
[0013] FIG. 1B illustrates an exemplary cell monolayer formed in an
exemplary sample container consistent with the disclosed
embodiments;
[0014] FIG. 1 C illustrates an exemplary cell monolayer formed in
an exemplary sample container consistent with the disclosed
embodiments;
[0015] FIG. 2A illustrates the blocking and diverting effect on the
electric current flow by a spherical insulator cell;
[0016] FIG. 2B illustrates the effective surface for
electroporation on a cell;
[0017] FIG. 3 illustrates the effect of cell size on the effective
electroporation surface;
[0018] FIG. 4 illustrates three representative neighboring cell
positions with the indicated direction of the electric current;
[0019] FIG. 5 illustrates the distribution of the electric current
that flows through a cell monolayer;
[0020] FIG. 6 illustrates the use of cell-mimicking artificial
insulator particles in boosting up the total cell number;
[0021] FIG. 7 illustrates an exemplary sample container consistent
with the disclosed embodiments;
[0022] FIG. 8 illustrates an exemplary sample container consistent
with the disclosed embodiments;
[0023] FIG. 9 illustrates an exemplary sample container consistent
with the disclosed embodiments;
[0024] FIG. 10 illustrates an exemplary use of centrifugation in
making a compact cell monolayer or multiple monolayers of cells for
electroporation or electrical cell fusion consistent with the
disclosed embodiments;
[0025] FIG. 11 illustrates an exemplary sample container consistent
with the disclosed embodiments;
[0026] FIG. 12 illustrates an exemplary process of electroporation
consistent with the disclosed embodiments;
[0027] FIG. 13A illustrates an exemplary sample container
consistent with the disclosed embodiments;
[0028] FIG. 13B illustrates an exemplary use of an exemplary lower
medium layer for electroporation consistent with the disclosed
embodiments; and
[0029] FIG. 14 illustrates an exemplary capillary assisted
electroporation.
DETAILED DESCRIPTION
[0030] Reference will now be made in detail to exemplary
embodiments of the invention, which are illustrated in the
accompanying drawings. Wherever possible, the same reference
numbers will be used throughout the drawings to refer to the same
or like parts.
[0031] FIG. 1A illustrates an exemplary electroporation apparatus
100 consistent with the disclosed embodiments. The apparatus 100
includes a sample container 10. The sample container 10 includes an
insulator chamber 14, a first electrode 15a, and a second electrode
15b. Within the sample container 10, an interface is formed on the
surface of a lower medium layer 12 and below an upper medium layer
13. A cell monolayer 11 across the electric current field may be
formed on the interface. A cell monolayer, as used in this
disclosure, refers to a single, compactly packed layer of cells. A
cell monolayer is therefore sometimes referred to as a compact cell
monolayer, or a compact monolayer. The apparatus 100 also includes
a pulse generator 18. The sample container 10 may be placed in the
pulse generator 18, which delivers an electrical pulse through the
first electrode 15a and the second electrode 15b. FIG. 1B provides
a cross section view of the cell monolayer 11 within the insulator
chamber 14. As shown in FIG. 1B, the monolayer 11 occupies the
cross section area of the insulator chamber 14.
[0032] Apparatus 100 may be implemented using certain concepts for
modeling cell electroporation. FIG. 2A illustrates the effect of a
spherical cell with the radius R on the electric current flow or
electric field that was originally uniform. For a typical
electroporation such as delivery of DNA, RNA or proteins to cells,
the electric shock takes place in a medium or other saline buffer
solutions. Compared to extracellular solution and intracellular
cell plasma, the lipid-bilayer based cell membrane has much lower
electric conductance and most of the electric current bypasses the
interior of the cells. A cell is thus similar to an insulator
object.
[0033] The insulator effect of the cell membrane protects the cell
interior from a short-time exposure of strong electric field during
electroporation. As shown in FIG. 2A, the blockade and diversion of
the electric current by an insulator-like spherical cell changes a
uniform electric field to one that is bulged around the cell.
[0034] A local point on the cell membrane can be designated by its
radius angle .theta. from the direction of general electric
current. The negatively charged molecules such as DNA, RNA and
proteins in a conductive medium move in the opposite direction to
that of the electric current.
[0035] For a single cell with a radius of R placed in an originally
uniform electric-current field, the transmembrane potential at a
given point on the membrane with a radius angle .theta. can be
roughly modeled by the equation
V.sub..theta.=1.5E.sub.0Rcos .theta., (I),
where E.sub.0 is the field strength of the original uniform
electric field. When .theta. equals to 0.degree. or 180.degree. at
the two topical points relative to the direction of the overall
electric field, cos .theta. equals to 1 or -1 and the transmembrane
potential value is the highest. At the topical point downstream of
electric current (.theta.=0.degree.) but not upstream of electric
current (.theta.=180.degree.), negatively charged molecules such as
DNA, RNA and proteins pass through the membrane under the greatest
electrical potential. On the contrary, the transmembrane potential
is zero at the points on the cell membrane where .theta. equals
90.degree., although the electric current is strongest just outside
these membrane points.
[0036] The transmembrane potential is largest when .theta. is
0.degree. and decreases to zero potential when .theta. is
90.degree.. A larger transmembrane potential at a local membrane
point can produce a larger force to transport molecules. To deliver
a substance, a minimal transmembrane potential V.sub.min would be
required. A maximal value of .theta., or .theta..sub.max could be
reached between 0.degree. and 90.degree. where the transmembrane
potential becomes V.sub.min. Meanwhile, the transmembrane potential
on cell points with smaller .theta. cannot be higher than the
potential that could irreversibly damage the cell. The
.theta..sub.max defines the largest effective electroporation
surface.
[0037] FIG. 2B illustrates the effective electroporation surface
(represented by the shaded area) on a spherical cell. The effective
electroporation surface, or the effective surface for
electroporation, as used in this disclosure, refers to the portion
of cell surface that has sufficient transmembrane potential to
allow exogenous substances, such as DNA, RNA, or proteins, to enter
the cells. In an electroporation to introduce large molecules such
as DNA, RNA or proteins to the cells, the cells can survive only
under certain transmembrane potential. In FIG. 2B, V.sub.max
represents the maximally tolerable reversible transmembrane
potential, above which a cell would be irreversibly damaged.
V.sub.min represents the minimally permeable transmembrane
potential that allows effective electroporation, below which the
exogenous substance cannot enter the cell. Both V.sub.max and
V.sub.min are determined by the membrane characteristics. V.sub.max
would be the same regardless of the kind of target substance to be
delivered, whereas V.sub.min is related to the target molecular
properties such as size and electric charge. Larger molecules would
probably have larger V.sub.min for delivery. The window between
V.sub.max and V.sub.min, which is the effective range of the
transmembrane potential to electroporate the cell, may be small
especially for delivering larger molecules.
[0038] As shown in FIG. 2B, only the topical point of the cell can
reach V.sub.max, the highest transmemberane potential. The outer
boundary of the shaded effective electroporation surface has the
transmembrane potential of V.sub.min and the radius angle of
.theta..sub.max. For negatively charged molecules such as DNA, RNA
and proteins, the effective electroporation surface is located
downstream of the electric current.
At the topical point, [0039] .theta.=0.degree. and cos
.theta.=1,
[0039] giving
V.sub.max=1.5E.sub.0R.
At .theta..sub.max where transmembrane potential decreases to
V.sub.min,
V.sub.min=1.5E.sub.0Rcos .theta..sub.max=V.sub.maxcos
.theta..sub.max.
Therefore .theta..sub.max is determined by satisfying
cos .theta..sub.max=V.sub.min/V.sub.max.
[0040] According to this modeling, the topical point has the
highest rate of effective molecular transport. The local
transportation rate decreases with the increase of .theta. until it
becomes zero at .theta..sub.max.
[0041] When individual cells of different radius are placed in a
uniform electric field, each cell has a different transmembrane
potential profile. The absolute values of V.sub.min and V.sub.max
are subject to certain variations according to circumstances. For
example, when different electrical pulse shapes such as exponential
decay wave or square wave are used, V.sub.min and V.sub.max values
might be different. However, the ratio of V.sub.min/V.sub.max is
probably not as sensitive to these types of alterations.
[0042] FIG. 3 illustrates how cell size affects the effective
electroporation surface. The cell membrane is essentially a lipid
bilayer dotted with membrane proteins including some channels.
Cells of the same type have similar membrane compositions although
the cell size varies to some extent. Therefore, electrical
properties of the membrane such as V.sub.max and V.sub.min may be
considered identical on different local point of a cell and for the
cells of the same type but of different size. Even for different
cell types, many mammalian cells probably share similar membrane
electric properties including V.sub.max and V.sub.min since the
membranes are essentially a lipid bilayer similarly dotted with
different proteins.
[0043] As shown in FIG. 3, three individual free cells, a large one
on the left with the radius "R", an intermediate one in the middle
with the radius "r" and a small one on the right are analyzed. The
three cell centers are aligned so that the circles can represent
either the physical cells or transmembrane potential profiles. The
electric field strength is set for the large cell to reach
V.sub.max at the topical point, and the effective electroporation
surface is shaded between the topical point with V.sub.max and the
outer boundary with V.sub.min. When the large cell obtains optimal
electroporation,
V.sub.max=1.5E.sub.0R
giving
E.sub.0=V.sub.max/(1.5R).
The intermediate cell (radius r<R) would have a lower
transmembrane potential and at the topical point. The transmembrane
potential V.sub.top at the topical point is given as
V.sub.top=1.5E.sub.0r=V.sub.maxr/R.
The effective surface for the intermediate cell with transmembrane
potential larger than V.sub.min is defined by the outbound angle
.theta..sub.r satisfying
V.sub.min=1.5E.sub.0rcos .theta..sub.r=V.sub.topcos
.theta..sub.r=(V.sub.maxr/R)cos .theta..sub.r,
giving
cos .theta..sub.r=(V.sub.min/V.sub.max)(R/r).
The effective electroporation surface on the intermediate cell is
smaller than that of the large cell and it is shaded between the
topical point with V.sub.top and the outer boundary with V.sub.min.
When r/R=V.sub.min/V.sub.max, cos .theta..sub.r becomes 1 and the
effective surface diminishes to zero. Therefore the minimum radius
of cell to obtain effective electroporation is
r.sub.min=(V.sub.min/V.sub.max)R.
The small cell shown on the right with a radius less than r.sub.min
would not have any point reaching V.sub.min and there is no
effective electroporation surface.
[0044] Therefore, cell size is an important factor in
electroporation. Larger cells not only have higher transmembrane
potential at the topical point but also larger effective surface.
When a higher electric current field is applied so that the
transmembrane potential of a smaller cell can reach V.sub.max, a
larger cell may not be able to survive. The difference in cell size
is unavoidable and accounts for some heterogeneity of cellular
properties in electroporation. For example, if 95% of a cell
population has a radius variation of about 20% with normal
(Gaussian) distribution, and V.sub.min/V.sub.max (i.e.,
r.sub.min/R) is about 90%, only the cells within a range of a
radius variation of about 10% may be effectively electroporated.
Based on Gaussian distribution, the highest theoretical
electroporation efficiency is about 67.3% when the midsized cells
are electroporated. For larger molecules, V.sub.min/V.sub.max
(i.e., r.sub.min/R) is about 95%, the highest theoretical
electroporation efficiency becomes about 37.6%.
[0045] The cell size problem also extends to different cell types.
Since many types of mammalian cells would have similar V.sub.min
and V.sub.max, cell types of smaller sizes require much higher
electric current field strength to reach V.sub.min and often the
toxicities related to high electric current such as heat, free
radicals, gas and metal ions may irreversibly damage the cells
before they are effectively electroporated. Cell size cannot be
easily changed, so an electroporation method that accommodates the
variability in cell size is desirable but not currently
available.
[0046] The above analysis of the transmembrane potential of
individual ideal cells lays down the foundation for understanding
cell electroporation. In an electroporation, it is often desirable
to use a large number of cells in the order of 10.sup.6 to
10.sup.7. The cells are also crowded in a small volume mainly for
two reasons: i) to achieve a high concentration of a target
substance to be delivered, ii) a smaller sample volume requires
less energy and therefore the pulse generator is easier to
manufacture.
[0047] In a typical electroporation with 10 million cells suspended
in 0.2 ml medium, each cell occupies an average medium space of
20,000 cubic microns (.mu.m.sup.3). Each cell occupies a space
equivalent to a cube with the side length of about 27 .mu.m, not
much larger than the diameter of typical mammalian cells. Therefore
in a typical electroporation, the average distance between cells is
comparable to the cell diameter of most mammalian cells, i.e.,
cells may be very close to each other.
[0048] The Equation (I) is valid only when a single free cell is
placed in a uniform electric field. The Equation (I) can be roughly
applied when a small number of cells are placed in a uniform
electric field with the distance between them far exceeding the
cell diameter. In an electroporation when cells are crowded, the
electric field surrounding each cell is shaped by this cell itself
and other cells in its proximity. While the inherent membrane
properties V.sub.min and V.sub.max are still the same, the Equation
(I) can no longer be applied to calculate the transmembrane
potential at a given point. As a result, the profile of the
electric field becomes very complicated and unpredictable. The
random positioning of cells constitutes yet another layer of
heterogeneity in electroporation efficiency of cells even if the
cells are perfectly equal in size.
[0049] Thus, an analysis of the complex electric field is presented
in order to better understand the electric field and to facilitate
the design of improved methods of electroporation and electrical
cell fusion.
[0050] FIG. 4 illustrates three types of representative cell
positioning with cells of an equal size. The first type is a free
cell (F) not affected by neighboring cells. The second and third
types represent two special ways of positioning neighboring cells.
In the first special positioning, two or more cells closely line up
in a longitudinal fashion along the direction of general electric
current flow. This positioning is represented by cells S1, S2 and
S3. In the second special positioning, cells are closely arranged
laterally on a cross-sectional plane that is substantially
perpendicular to the direction of general electric current flow.
This positioning is represented by cells E1, E2 and E3. While it is
more complex to derive the exact equations of the transmembrane
potentials on these cells, a qualitative analysis can be done in a
relevant and sufficient manner for developing improved methods of
electroporation.
[0051] As shown in FIG. 4, S1, S2 and S3 are closely lined up and
they do not block and divert the flow of electric current
significantly more than the free cell F. Therefore, only S1 and S3
have a similar transmembrane potential profile on the topical
surface to that of the cell F. Moreover, only the topical surface
of the S1 cell downstream of the electric current (the shaded area
in FIG. 4) would be effective for electroporation of negatively
charged molecules. The S3 cell can be like the S1 cell for
electroporation of negatively charged molecules if the direction of
electric current is reversed. S2 cell would have lower
transmembrane potential because of the shielding effect from S1 and
S3 cells. The effect of a leading cell shielding the following
cells from obtaining electroporation is defined as the longitudinal
shielding effect. The longitudinal shielding effect becomes less
prominent as the distance between cells increases or when the
lineup deviates from the strict longitudinal orientation. The
longitudinal lineup thus introduces a level of undesirable
heterogeneity in electroporation by decreasing the efficiency of
electroporation for many shielded cells.
[0052] On the contrary, the cells E1, E2 and E3 collectively exert
a much bigger effect in blocking and restricting the flow of
electric current. As a result, E1, E2 and E3 would have higher
transmembrane potentials than that of the cell F just as if they
formed an extra-large individual cell. The laterally arranged cells
would no longer follow the transmembrane potential Equation (I).
Because there is less overall electric current near the plane of
cells, the transmembrane potential would decrease from the topical
point more gradually than that of a free individual cell. In a
qualitative description, each of the E1, E2 and E3 cells would have
a larger effective electroporation surface than an F cell for an
enhanced effect of electroporation between the points of V.sub.max
and V.sub.min, although the shape of the effective surface may
become somewhat irregular.
[0053] While a three-dimensional contour map would describe the
actual effective electroporation surface on E1, E2 and E3 more
precisely, a two-dimensional shading as used in FIG. 4 suffices to
roughly illustrate the increased effective surface area. The effect
of laterally arranged cells enhancing each other's electroporation
accessibility is defined as the lateral enhancing effect. The
lateral enhancing effect becomes less prominent as the distance
between cells increases or when the cells deviate from the
cross-sectional planar positioning. Overall, these cells can be
rated for electroporation accessibility at the indicated electric
current direction as follows:
E2,E1,E3>S1,F>S2,S3
[0054] In a typical electroporation carried out in cell
suspensions, the complex cell-to-cell electrical interactions can
be characterized in three main categories: longitudinal shielding,
lateral enhancing and hybrid interaction of longitudinal shielding
and lateral enhancing. The hybrid interactions are between
neighboring cells in a position that is neither predominantly
longitudinal nor predominantly lateral and these interactions have
less shielding or enhancing effect. The qualitative analysis
revealed that longitudinal shielding is usually undesirable for
achieving a high efficiency in electroporation. The effect of
longitudinal shielding in a cell suspension is hard to avoid.
Alternating the direction of electric current may present both
topical surface of a longitudinal lined up cells for increased
efficiency of electroporation. The lateral enhancing effect is
beneficial for electroporation and especially helpful for difficult
cell types of small sizes that require a destructively high
electric field strength to reach V.sub.min.
[0055] Based on these understandings, it becomes desirable to
eliminate or diminish the undesirable longitudinal shielding effect
and maximize the effect of lateral enhancing during
electroporations.
[0056] Returning to FIG. 1A, when cells reside in the compact
monolayer 11 across the electric current field, the longitudinal
shielding effect is geometrically eliminated and the lateral
enhancing effect is increased between cells.
[0057] As shown in FIG. 1A, the insulator chamber 14, the first
electrode 15a, and the second electrode 15b seal the upper medium
layer 13 and the lower medium layer 12 within the sample container
10 so that the sample would not leak. The container 10 may be in
different shapes. For example, the container 10 may be cylindrical
or non-cylindrical.
[0058] The insulator chamber 14 used in the sample container 10 may
be made of nonconductive materials such as plastics, rubber,
polystyrene, polypropylene, polyethylene, polycarbonate,
polymethylmethacrylate, polyimide, polydimethylsiloxane, cyclic
olefin copolymer, thermoplastic polyester elastomer, glass, quartz
and silicon. The insulator chamber 14 may be made of one or more
types of materials so that it can be strong and fit tightly with
the electrodes.
[0059] The first electrode 15a and the second electrode 15b may be
made of conductive materials such as aluminum, iron, steel, nickel,
titanium, zinc, copper, tin, silver, graphite and alloys. They can
also be made of gilded metals, surface-modified metals or
nonconductive materials such as rubber or plastics coated or
intermixed with conductive materials. The first electrode 15a and
the second electrodes 15b may be made transparent for microscopic
observation of cells using materials such as indium-tin oxide,
aluminum-doped zinc oxide and antimony-doped tin oxide.
[0060] The electrode 15a may be an upper electrode, and the
electrode 15b may be a lower electrode. The distance between the
two electrodes 15a and 15b is preferred to be larger than 1 mm for
easy handling of the liquid, and less than 50 mm to avoid consuming
too much target substance to be delivered. In one embodiment, a
distance between the two electrodes 15a and 1b is ranged from 1 mm
to 30 mm for easy handling and conservation of reagents. The shape
and dimension of the electrode plates 15a and 15b may be determined
according to those of the container 10. Since the electric power
used in the monolayer method is very low as discussed in the
paragraphs [0071] to [0075] and little amount of metal ions is
released, it is usually not necessary to use precious metals such
as gold or platinum to make the electrodes 15a and 15b. However,
precious and inert metals such as gold and platinum may be used to
make the electrodes 15a and 15b when cost is not a concern or when
the containers need to be reusable.
[0061] The pulse generator 18 generates an electrical pulse for
electroporation of the biological cells. The generator 18 may
generate one or several different pulse forms such as, exponential
decay wave, square wave or rectangular wave, high-frequency waves,
and a combination of multiple wave forms. The pulse forms for
electroporation may be predetermined based on cell type, the type
of the container, and/or other data. The pulse generator 18 may
thus be programmed to deliver the predetermined pulse form for the
electroporation. In this disclosure, a pulse, or a pulse form, may
refer to a single pulse or a combinatorial pulse composed of
multiple pulses or pulse forms.
[0062] The compact cell monolayer 11 can be formed on the surface
of an electrode or anywhere between the two electrodes. To form a
cell monolayer 11 that is not directly on an electrode, an
interface can be made between the two conductive medium layers 12
and 13 for cells to stay on. A cell suspension containing an
appropriate number of cells is placed on the surface of the lower
medium layer 12. The cell suspension may be formed by suspending
the cells in a medium or buffer used to form the upper medium layer
13 or other appropriate medium or buffer. The cells may settle to
the interface between the lower medium layer 12 and the upper
medium layer 13 by natural gravity or an artificial centrifuge
force. The pulse generator 18 delivers a certain form of electrical
pulse to the cell monolayer through the first electrode 15a and the
second electrode 15b and the pulsing takes place after the cell
monolayer is formed.
[0063] A stable interface needs to be maintained between the upper
medium layer 13 and the lower medium layer 12 during
electroporation. The upper medium or buffer layer 13 usually comes
from the medium or buffer used to create the cell suspension or any
other appropriate medium or buffer. The medium or buffer used in
the upper medium layer 13 may be any suitable medium or buffer,
such as MEM, DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution.
The lower medium or buffer layer 12 may be a solution with a higher
density such as solutions containing sugars, glycerol, polyethylene
glycol (PEG) and ficoll. The medium or buffer used in the lower
medium layer 12 may be any suitable medium or buffer, such as MEM,
DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, which may or
may not be the same to the medium or buffer used in the upper
medium layer 13. The lower medium layer 12 may also be formed from
a semi-solid gel such as agar or agarose based gel, silicone gel,
polyacrylamide gel, collagen or gelatin gel, matrigel, hyaluronic
acid gel, alginate gel, polyethylene glycol gel, methyl cellulose
or other modified cellulose based gel, acrylates gel, polyglycols
gel and propylene glycol gel.
[0064] Furthermore, the lower medium layer 12 may be formed from a
porous solid matrix doused with a medium or buffer. The solid
matrix is porous and preferably hydrophilic so that conductivity of
the lower medium layer 12 is maintained. The solid matrix may be
made of materials such as silicone, resins, glass fibers,
polymethacrylates, silicates, modified cellulose, polyvinyls,
polylysine, polyacrylic acid, polyethylene glycol, polyacrylamides
and co-polymers. Certain materials that form the lower medium layer
12 may falls between the physical states of liquid and semi-solid,
or semi-solid and solid, or liquid and solid. Such physical state
of the lower medium layer 12 would not affect electroporation as
long as a stable interface may be formed and maintained between the
upper medium layer 13 and the lower medium 12. The target molecules
to be delivered may be in the upper medium layer 13, or the lower
medium 12, or both.
[0065] The lower medium layer 12 that is semi-solid or solid may be
pre-formed in the sample container 10. For example, an agarose gel
in suitable medium, such as RPMI medium, may form the lower medium
layer 12 and may be precast in the sample container 10.
[0066] Usually, two medium layers, an upper medium layer 13 and a
lower medium layer 12 would be sufficient to form the cell
monolayer 11 for electroporation. However, in certain embodiments,
more than two conductive medium layers may be used as long as there
is at least one medium interface for cells to stay on. Sample
containers of different cross section area may be made to form
interfaces with different sizes to accommodate different number of
cells.
[0067] FIG. 1C illustrates an exemplary sample container consistent
with the disclosed embodiments. As shown in FIG. 1C, the cell
monolayer 11 is formed directly on the electrode 15b. To form the
monolayer 11, a cell suspension with an appropriate number of cells
in an appropriate medium or buffer is loaded in the sample
container. The cells settle to the electrode 15b to form the
monolayer 11 either under natural gravity or by centrifugation.
This is suitable when the electric field strength required is low
and/or the cells may tolerate the toxicities.
[0068] For electroporation of most eukaryotic cells, the medium or
buffer that forms the two medium layers 12 and 13 usually contains
salts to maintain a proper osmotic pressure. The salts in the
medium or buffer also render the medium layers 12 and 13
conductive. For electroporation of very small prokaryotic cells
such as bacteria, sometimes water is used as a low conductance
medium to allow a very high electric field strength. In that case,
the charged molecules to be delivered still render water based
medium more conductive than the lipid based cell membranes and the
medium may still be roughly considered as conductive especially
compared to cell membranes.
[0069] The number of cells to be used to form the monolayer 11 is
determined by the area of the surface where the monolayer 11 is
located and the area occupied by an average sized cell. The cell
concentration to be used may be empirically determined by
observation of the monolayer 11 under microscope in another
transparent container with a known area, by electrical resistance
measurement or by testing of electroporation efficiencies with
different cell numbers. Many mammalian cells may need a density of
0.2-2 million cells per square centimeter for a compact monolayer.
The properties of different cells such as average area of occupancy
and preferred electric field strength can be stored in a database
for easy reference. Sample containers of different interface area
can be made to accommodate different cell numbers.
[0070] Two or more monolayers of cells may stack up compactly on
the medium interface and the resulted cell pellet can still be
electroporated. In this situation, the electroporation may become
more heterogeneous with increased variation of molecular transport
among cells with the exception of a cell double layer. In a cell
double-monolayer, the two monolayers of cells can still have quite
homogeneous electroporation when an alternating current pulsing
scheme is used. A pellet, as used in this disclosure, refers to a
group of cells that form more than a single, compactly packed
layer. The cells in a pellet may or may not form discernible
layers.
[0071] Multiple monolayers of cells, also referred to as a cell
pellet, can be conveniently used in applications that require a
very high cell number and that the variation of molecular transport
among cells is not a major concern. The multiple-monolayer method
of electroporation can be considered as a special form or an
extension of the monolayer electroporation method. Compared to
traditional electroporation with a cell suspension, the
multiple-monolayer method of electroporation is still advantageous
in efficiency, cell survivability and cost effectiveness. The
methods and devices described for a cell monolayer are all
applicable to cells in multiple layers or in a pellet.
[0072] The monolayer electroporation method substantially decreases
the necessary overall electric field strength. FIG. 5 illustrates
the distribution of the electric current that flows through the
cell monolayer 11. In FIG. 5, the three-dimensional distribution of
the electric current around a cell monolayer is illustrated by a
simplified two-dimensional representation.
[0073] Because cells are similar to insulators, most of the
electric current that goes through the compact cell monolayer 11
would go through the crevices between the cells. Only the electric
current field strength within the crevices between the cells would
be similar to what is needed for a traditional electroporation in a
suspension. In other locations, the electric current is spread to a
very low level.
[0074] Just outside the monolayer 11, the electric current density
or the electric field strength is dispersed to a very low level,
thus creating an enlarged region of low electric field (L-F) near
the topical surfaces of the cells. Within the L-F regions, there is
very little electric potential change regardless of the variation
in cell sizes and crevice sizes, as long as the monolayer is
compact and the crevices are relatively small. The shaded areas
represent effective electroporation surfaces for negatively charged
molecules at the given direction of electric current.
[0075] This is beneficial for cell survival since the space of the
crevices are small and the effect of small regions of stronger
current would quickly dissipate. A crevice between cells may have
an irregular shape, therefore the width or the area of a crevice
would not be uniform. The crevice width can be roughly defined as
the average width of a electric current path below the effective
electroporation surfaces.
[0076] For example, when the cells in the monolayer 11 occupy 80%
of the space and leave about 20% the total crevice space between
cells, the total electric current would be about 20% of what is
required to generate a similar transmembrane potential for a
suspension of these cells in the same container. The overall
increase of electric resistance would be small, as the resistance
of the monolayer 11 is about 5 cells' depth of medium (reciprocal
of 20% is 5) and this is small compared to the usual distance
between electrodes. This translates into about 20% of the voltage
and only about 4% of the required power (20%.times.20%) for
traditional electroporation in the same container. If the space
between cells is 10% of the total space, the voltage required would
be about 10% and the power required would be about 1%, and so
on.
[0077] As used in this disclosure, the term "compact" refers to the
extent that the cells occupy the monolayer area. The term
"compactness," refers to the percentage of the cell monolayer area
that is occupied by the cells. A reasonable minimal working
compactness for the monolayer 11 would be around 50% to take
advantage of the lateral enhancing effect. As the layer compactness
increases, the lateral enhancing effect becomes more significant
and beneficial. Complete compactness is reached when no more cells
can fit into the monolayer at a given gravity or centrifugal force.
At complete compactness, the total space between cells would be
small but not totally eliminated. When cells are compactly
arranged, they may not appear circular or spherical because of
their plasticity. Slightly going over a complete compactness in the
monolayer would cause a small decrease in efficiency, as a small
portion of cells would lie on top of each other and introduce some
undesirable longitudinal shielding effect.
[0078] Compared to free individual cells in a suspension, every
cell in the compact monolayer 11 would have a larger effective
surface. Within the effective surface, the transmembrane potential
does not drop from the topical point as steeply as free individual
cells. In traditional electroporation in a suspension, cells have
heterogeneity in both transmembrane potential and effective
delivery surface area. However, cells of variable size would have
similar transmembrane potential in a compact monolayer. This is
because overall current on both sides of the monolayer are small
and therefore the potential is substantially equal just outside of
the monolayer. A smaller cell in the monolayer 11 would have only
smaller effective delivery surface, but almost equal transmembrane
potential compared to larger cells.
[0079] With the monolayer method, the amount of target molecules
delivered to each cell would be much less variable. For example, a
10% cell diameter difference may cause a difference greater than an
order of 10 in substance delivery in traditional electroporation.
By contrast, the variation in substance delivery may be about 10%
in compact monolayer electroporation. Because of the more leveled
electric potential profile on the topical surfaces, the working
voltage range (percentage wise) is increased compared to a similar
pulsing scheme in traditional methods.
[0080] Because low electric power is used in monolayer
electroporation, many of the cell toxicities related to electric
current and electrodes would be reduced. Directly arranging the
compact monolayer 11 on the electrode 15b is relatively simple and
it might be suitable for cells that require very small electric
field strength in a certain buffer or for cells that can tolerate
the toxicities. Using an interface between the conductive medium
layers 12 and 13 may be advantageous, because physically keeping
the cells away from the electrodes 15a and 15b is a very effective
way of avoiding electrochemical toxicities to the cells. A
conductive medium or buffer conducts electricity by solute ions and
it is distinct from an electrode that typically conducts
electricity by free electrons.
[0081] Also because the lower electric power is used in monolayer
electroporation, it becomes easier to manufacture the pulse
generator 18 for monolayer based electroporation method compared to
making pulse generators for traditional electroporation methods.
When the output power is lower, it is easier to generate different
pulse forms. One type of common pulse form is the exponential decay
wave, typically made by discharging a loaded capacitor to a sample.
The exponential decay wave can be made less steep by linking an
inductor to the sample so that the initial peak current can be
attenuated. Another type of common pulse form is square wave or
rectangular wave. Other waveforms such as high-frequency waves can
also be easily generated when desired. A single waveform or
multiple waveforms in a sequence can be applied to a sample.
[0082] When multiple waveforms in a specified sequence are used,
they can be in the same direction (direct current) or different
directions (alternating current). Using alternating current can be
beneficial in that two topical surfaces of a cell instead of just
one can be used for molecular transport. Especially for
electroporation of cells packed in a multiple-monolayer pellet, an
alternating current pulsing scheme can alleviate the longitudinal
shielding effect as explained in the description for FIG. 4. The
pulse generator can be controlled by a digital or analog panel.
Further, the pulse generator can include a rechargeable battery so
that the unit can become cordless when desired.
[0083] The monolayer method is beneficial to cells of different
sizes. The cells of smaller sizes are more benefited because of
their requirement for higher electric field strength in a
traditional electroporation. The monolayer method may be
conveniently applied to cells that typically grow in suspensions,
such as hematopoietic cell lines, lymphocytic cell lines and cells
of blood origin.
[0084] For adherent cells that usually grow on some supporting
substance or structure, they may be temporarily suspended for
electroporation. The adherent cells may be removed from their
supporting structure by common means such as mechanical dispersing
and trypsin treatment. The adherent cells then may be suspended in
appropriate medium or buffer and form the monolayer for
electroporation.
[0085] The adherent cells may also be cultured on the interface
between two medium layers 12 and 13 inside the sample container 10.
The cultured adherent cells within the sample container 10 may be
directly electroporated. However, it is more difficult to control
the quality of such electroporations for several reasons. First,
the cell surface area variation of adherent cells is much larger
than suspension cells and the electrical properties of cells are
more variable. Second, it is hard for adherent cells to cover the
monolayer 11 uniformly. Cells may be absent in some devoid areas
and may be stacked in some crowded area. The cells close to the
devoid areas may be subject to increased electrical toxicity. The
electroporation efficiency may be decreased in a crowded area
because some cells are on top of other cells. Third, the
electroporation may only be performed during a specific time when
cells reach confluence but not over growing. As a result, the time
to perform the electroporation is restricted. Fourth,
reproducibility of results can be lower because the time needed for
cell culture introduces variation among samples.
[0086] If the cell number is low, cell-mimicking artificial
insulator particles with a similar diameter may be used to boost up
the total cell number. FIG. 6 illustrates the use of artificial
insulator particles in electroporation of cells in low numbers. The
insulator particles 16 (shaded) are randomly mixed with real cells
17 (open circles). The mixed cell monolayer 11 is formed on the
interface of two conductive medium layers 12 and 13. The medium and
the cells are contained in an insulator chamber 14 and the
electrical pulse is delivered through the electrodes 15a and
15b.
[0087] The insulator particles 16 may help form a compact monolayer
11 and restrict the electric current flow as the real cells 17 do.
When the number of the insulator particles 16 used exceeds the
number of cells in large quantity, the exact number of cells 17
becomes unimportant and the same number of insulator particles 16
may be used with samples containing different number of cells 17,
thus simplifying the procedure.
[0088] The size of the insulator particles 16 is generally similar
to that of the cells 17. It is acceptable that the difference
between the diameter of insulator particles 16 and that of the
cells 17 is within an order of 10.
[0089] The insulator particles 16 may be made of or coated with
materials having certain biological properties so that they may be
left with the cells 17 after electroporation. The insulator
particles 16 may also be made of materials having magnetic
properties so that they may be separated by a magnetic method.
Other methods of separating the insulator particles 16 from the
cells 17 may be based on differential rate of sedimentation or
differential density. In certain embodiments, the artificial
insulator particles 16 may be other types of real cells, such as,
cells that may allow easy separation after electroporation and
cells that may be irradiated or drug-treated to lose the cell
viability or to stop the cell growth.
[0090] The sample container 10 as shown in FIG. 1A is one exemplary
container consistent with the disclosed embodiments. Other types of
sample containers may also be used.
[0091] FIG. 7 illustrates an exemplary sample container 20
consistent with the disclosed embodiments. The sample container 20
includes a fixed electrode 25, a movable electrode 26 that also
functions as a sealing cover, a cylindrical insulator chamber 21,
an excess-receiving groove 24.
[0092] The chamber 21 has an open groove at the bottom end for
fixing the electrode 25 and another open groove at the top of the
chamber wall to receive the movable electrode 26. The diameter of
the electrode 25 may be slightly larger than the diameter of the
groove at the bottom end of the insulator chamber 21 so that the
electrode 25 may be tightened by the tension generated in the
bottom end of the insulator chamber 21. Alternatively the electrode
25 may be fixed to the insulator chamber 21 for sealing of the
container bottom by gluing or any other appropriate methods.
[0093] The movable electrode 26 is embedded in an open insulator
cover 22 that is connected to the main insulator chamber 21 through
a flexible linkage 23. The cover 22 may fit in the top groove in
the wall of the main insulator chamber 21 tightly, allowing the
electrode 26 to cover on an inner rim in the main insulator chamber
21 and seal the sample. To securely seal the medium layers 12 and
13 containing the cell monolayer 11 without air bubbles, the volume
of the cell suspension to be added can be slightly larger than the
allowed volume in the sealed container 20 so that there is a little
excess liquid to ensure a perfect sealing. The excess liquid pushed
out by closing down the electrode cover 26 can flow to the
excess-receiving groove 24 etched at the top of the wall of the
main insulator chamber 21. After electroporation, the cells in the
excess liquid may be discarded as they are not electroporated.
[0094] While it is good practice to seal the sample inside the
container without air bubbles, some small air bubbles can actually
be tolerated during electroporation of cells in the monolayer 11 or
in multiple monolayers. Since the overall electric field strength
in the medium is very low, the disturbance of the electric field by
air bubbles near the electrode 26 would only leave a negligible
effect on the electric field near the cells on the medium
interface. Compared to traditional open cuvettes, a sealed
container may be advantageous because the air bubbles produced near
electrodes may be compressed and cause less disturbance to the cell
sample. A sealed container generally refers to a container that can
enclose a sample without an open side and that a liquid sample
inside would not flow around when the container is rotated to
different orientations.
[0095] A cylindrical chamber is relatively easy to manufacture, and
round-shaped electrode plates can easily fit in tightly even
without the use of any sealing glue. The cylindrical container 20
may be altered or include additional features. While the inside
space of the container 20 is cylindrical, the exterior of the
container may be in other shapes as long as the inside shape of the
container is maintained. The cylindrical container 20 may also be
made in different dimensions. In one embodiment, the distance
between the electrodes 25 and 26 may be between 1 mm and 50 mm. In
another embodiment, the distance between the electrodes 25 and 26
may be between 1 mm and 30 mm. The inner diameter of the main
insulator chamber 21 may be between 1 mm and 100 mm.
[0096] While cylindrical containers are convenient to manufacture
and use, a container 20 for monolayer electroporation may use any
suitable shape as long as the cells can form a monolayer on the
medium interface or the electrode 25. For example, a rectangular
container or container with other shapes would be suitable. A
snap-on closing mechanism or other closing mechanism may be used on
the rectangular container.
[0097] As shown in FIG. 7, the sealing cover includes the electrode
26 encased by the insulator cover 22. Other type of sealing cover
may also be used. For example, a sealing cover may be a properly
shaped conducting electrode 26 with a snap-on or screw-on closing
mechanism.
[0098] The insulator cover 22 may be snapped on to close the sample
container 20. A locking structure can be used to ensure a tight
closure. Other means such as screw-on type closing mechanism may be
used. For example, screw threads may be made on the open insulator
cover 22 and the main insulator chamber 21 for a tight closure. The
cover 22 may tightly fit to the inner rim of the main chamber 21 as
shown in FIG. 7, or it may fit on the main chamber 21 by the outer
rim. Optionally, a flexible linkage 23 may also be included for
convenient closure of the cover 22.
[0099] On each of these components of the exemplary sample
container 20, there may be markings or handles for convenient
handling. The insulator chamber 21 may be made of the materials
similar to those making the insulator chamber 14. The electrodes 25
and 26 may be made of the materials similar to those making the
electrodes 15a and 15b.
[0100] The present disclosure also contemplates a sample container
with open configuration. FIG. 8 illustrates an exemplary sample
container 30 consistent with the disclosed embodiments. As shown in
FIG. 8, the sample container 30 includes an insulator chamber 34, a
bottom electrode 35, a mesh-type electrode 36 with a matching metal
connector 37, and a sealing cover 39.
[0101] As shown in FIG. 8, cells form a monolayer 11 or multiple
monolayers on the interface between the lower medium layer 12 and
the upper medium layer 13. The mediums and the cells are contained
in the insulator chamber 34 of the container 30. The bottom
electrode 35 is fixed in a groove at the bottom end of the
insulator chamber 34. The container 30 may take any appropriate
shapes such as rectangle. The containers may be arranged in an
array for processing of multiple samples.
[0102] A mesh-type electrode 36 with a matching metal connector 37
is inserted to a groove at the top of the wall of the insulator
chamber 34. The mesh electrode 36 would allow free passage of the
cells and it is submerged in the upper medium layer 13 for
electroporation of the cells. The mesh electrode 36 may be inserted
before or after addition of the cell suspension. In addition, the
mesh electrode 36 may be fixed in the container 30 or removably
attached to the container 30. The mesh electrode 36 takes the same
shape of the container 30 to fit on the container 30. For example,
the mesh electrode 36 is rectangular when the container 30 is
rectangular.
[0103] The open configuration container 30 may be further protected
by a sealing cover 39 at the top if necessary. The sealing cover 39
may be made of plastic. Cells may be unloaded either after removing
the mesh electrode 36 or in presence of the mesh electrode 36.
[0104] The insulator chamber 34 may be made of the materials
similar to those making the insulator chamber 11. The electrodes
35, 36 and the connector 37 may be made of the materials similar to
those making the electrodes 15a and 15b.
[0105] The present disclosure also contemplates a sample container
with fixed electrodes and separate additional cover. FIG. 9
illustrates an exemplary sample container 40 consistent with the
disclosed embodiments. As shown in FIG. 9, the container 40
includes an insulator chamber 44, a first fixed electrode 45a, a
second fixed electrode 45b, and pre-formed semi-solid or doused
solid lower medium layer 12 within the container 40.
[0106] The insulator chamber 44 may be made of the materials
similar to those making the insulator chamber 14. The electrodes
45a and 45b may be made of the materials similar to those making
the electrodes 15a and 15b.
[0107] As shown in FIG. 9, a cell suspension fills up the gap
between the lower medium layer 12 and the first electrode 45a when
the container is placed vertically, i.e., the medium interface is
vertical. The container 40 is then immediately turned level so that
the medium interface becomes horizontal and the cells form the
monolayer 11 that settles on the interface between the lower medium
layer 12 and the upper medium layer 13. The cell suspension is
confined in the open-ended container 40 by natural surface tension
of the liquid and the cells may settle down by gravity but not a
strong centrifugal force. If a centrifugal force is desired, a
sealing cover (not shown in the drawing) may be used to seal the
medium layers 12 and 13 on the open side.
[0108] To arrange cells into a compact monolayer, preferably a cell
suspension containing an appropriate number of cells is placed on
top of the lower medium layer 12. Cells may settle on the interface
between the lower medium layer 12 and the upper medium layer 13
under the natural gravity or an artificial centrifugal force.
Alternatively, a lower medium with higher density can be added to
the cell suspension. The lower medium layer 12, the upper medium
layer 13, and the cell monolayer 11 may form under the natural
gravity or an artificial centrifugal force. It is simple and
low-cost to precipitate the cells to the interface by natural
gravity. On the other hand, with an artificial centrifugal force
stronger than gravity, a cell monolayer or a multiple-monolayer
pellet can be formed more quickly and more compactly.
[0109] FIG. 10 illustrates the use of centrifugation in making a
compact cell monolayer or a pellet of cells. The exemplary sample
containers 10 with the two electrodes 15a and 15b and the insulator
chamber 14 may be used to hold the two medium layers 12 and 13 and
the cell monolayer 11. One or more sample containers 10 can be
placed in one rotor with proper balancing. Other exemplary sample
containers, such as containers 20, 30, or 40, may be used in
centrifugation as well.
[0110] An exemplary centrifuge 50 consistent with the disclosed
embodiments is shown in FIG. 10. The centrifuge 50 includes a first
metal support 55, a second metal piece 56, an axis of rotation 58,
an electrical brush 59a and 59b, and proper in-rotor wiring.
Optionally, an in-rotor circuitry 54 may be included to generate an
electrical pulse in the rotor.
[0111] The first metal support 55 keeps the container in the rotor
and makes electrical contact with the bottom electrode 15b. The
second metal piece 56 is pressed by the centrifugal force to the
top electrode 15a to make an electrical contact. The two contact
points where the metal pieces 55 and 56 contact the sample
containers 10 may be wired to the axis of rotation 58 either as a
group or individually through the electrical brushes 59a and 59b on
the axis of rotation 58. When an in-rotor circuitry 54 is used, the
metal pieces 55 and 56 may be wired to the in-rotor circuitry 54.
The two metal pieces 55 and 56 thus function as conductor to
deliver an electrical pulse to the sample container 10.
[0112] The centrifuge 50 may be a swing-bucket centrifuge or a
fixed-rotor centrifuge. During centrifugation, the medium interface
or the cell monolayer 10 is substantially perpendicular to the arm
of rotation or the direction of centrifugal force as indicated by
an open arrow 57. The proper angle can be easily achieved in a
self-adjusting swing-bucket rotor or a fixed-angle rotor that
positions the medium interface in an electroporation container
nearly perpendicular to the arm of rotation. The axis of rotation
58 for a fixed-angle rotor can either be vertical or horizontal.
When the axis of rotation 58 is horizontal, the medium interface
needs to be substantially perpendicular to the arm of rotation.
[0113] Centrifugal force is proportional to the radius of rotation
and the square of angular velocity. For a typical rotation radius
of several centimeters to several decimeters, a rotation speed of
several hundred rpm (revolutions per minute) to several thousand
rpm is sufficient for most eukaryotic cells to form a cell
monolayer. Other rpm numbers may also be used. For small
prokaryotic cells such as bacteria, a rotation speed of several
thousand rpm may be needed. The time needed for centrifugation can
be from seconds to minutes. Other time may also be used. The
acceleration of rotation can be made gentle, so that cells do not
move sideways on the medium interface in a container. For a
fixed-angle rotor that positions the medium interface vertically,
rotation should start promptly so that cells in suspension do not
sink to one side to cause unevenness in cell distribution.
[0114] Theoretically, a flat medium interface in a rotor produces
slightly uneven centrifugal force because of the differential
rotation radius. This would not be a significant factor if the time
of centrifugation is not prolonged. A substantially even cell
distribution may be achieved through a longer rotation arm length,
a medium interface that reduces sideway cell movements and some
settling-down time before centrifugation. A sample container with
curved electrodes may be made. A curved medium interface may be
formed within the container that has equal radius of rotation on
all points. A container with a full-circle cylindrical medium
interface rotating around its own axis would ideally provide equal
radius of rotation on all interface points and it can use any
acceleration setting without problem of sideway cell movements.
[0115] As shown in FIG. 10, the metal pieces 55 and 56 may be wired
to the axis of the rotation 58 to provide an electrical pulse to
electroporate the cells on the cell monolayer 11 during
centrifuge.
[0116] An electrical pulse can be delivered after the cells are
brought into the monolayer 11 or a multiple-monolayer pellet. When
the centrifuge 50 is used, an electrical pulse can be delivered
after centrifugation or during centrifugation. To pulse after
centrifuge, a common laboratory centrifuges without in-rotor
electrical wirings may be used with suitable adapters for holding
the electroporation containers in the rotor. However, the sample
containers 10 need to be very carefully taken out of the centrifuge
50 to avoid disturbing the cell monolayer 10 or pellet before
pulsing. On the other hand, delivery of an electrical pulse during
centrifugation is advantageous in that the pulsing condition would
be more reliable.
[0117] After the delivery of an electrical pulse, cells can be
removed from the sample container 10 or remain in the container 10
if the medium for electroporation is also suitable for cell
maintenance. A centrifuge and a pulse generator can be integrated
into one machine, providing easy portability and convenient
control.
[0118] The carbon brush or electrical brush 59a and 59b can be used
to wire the final electrical pulse from the pulse generator
directly to samples in a centrifuge rotor. Alternatively, the final
electrical pulse for the samples can be generated by the in-rotor
circuitry 54 to avoid any signal noise from the electrical brushes
59a and 59b. The in-rotor circuitry 54 can be constructed near the
rotation axis 58 so that it is not subject to a high centrifugal
force. When the in-rotor circuitry 54 is used, the electrical
brushes 59a and 59b can be used to receive electrical energy and
control instructions and they are not in the final pulse delivery
loop. Contactless electrical power delivery can also be achieved
using magnetic energy transfer to avoid signal noise from the
electrical brushes 59a and 59b. When the in-rotor circuitry 54 is
used, wireless radio signals can be used to control the pulsing in
the rotor.
[0119] FIG. 11 illustrates an exemplary container 60 consistent
with the present disclosure. As shown in FIG. 11, the bottom
electrode 65 is enclosed in the insulator chamber 61. An insulated
metal wire 67 connects to the bottom electrode 65. The upper
electrode 66 encased by an open cover 62 can close onto the main
insulator chamber 61 and seal two medium layers 12 and 13 and the
cell monolayer 11. The cover 62 may be be linked the main chamber
by a linker 63. A groove 74 may be made in the main insulator
chamber to hold any excess sample. An electrical pulse is delivered
through the electrodes 66 and 67. This configuration may be useful
in preventing sample leakage during centrifugation.
[0120] In addition, the present disclosure may be used for
electrical cell fusion. When electrical cell fusion is the
objective of the electroporation, a double-monolayer configuration
with roughly two cell monolayers may be used in a sample containers
similar to the sample container used for electroporation, such as
the exemplary sample container 10, 20, 30, 40 or 60. Preferably,
each layer contains one type of cell, but mixing the two types of
cells are acceptable although the efficiency of fusion may be
lower.
[0121] The double-monolayer may be formed sequentially or together
by taking advantage of differential sedimentation speeds, i.e., one
type of cell can settle down first and the other one can follow
afterwards. A container with a mesh-type electrode may be
convenient for making two monolayers of different cells. The first
cell suspension may be added to a container just up to the mesh
electrode and the cells are collected onto the lower medium
interface. Then the second cell suspension may be added above the
mesh electrode so that they may be arranged evenly on top of the
first cell monolayer. Another possible way of making two sequential
monolayers is to use two cell suspensions in two different mediums
that can form an interface and the second cell suspension can be
added after the first cell suspension is already in a
monolayer.
[0122] The double-monolayer in suitable buffers can then be treated
by a suitable electrical pulse to promote cell fusion. The delivery
of electrical pulse may occur during centrifugation. Because there
is no target substance to be delivered to the cells during
electrical cell fusion, saving reagents is not an objective.
Therefore a sample container for cell fusion may have a larger
distance between the two electrodes so that it is easier to make
two monolayers of different cells sequentially. Furthermore, two
types of cell monolayers can form a sandwich of alternating cell
layers with three or more cell layers for electrical cell fusion by
repeating the steps of removing centrifuged medium and adding more
cell suspension.
[0123] FIG. 12 illustrates an exemplary process 200 for the
monolayer electroporation consistent with the disclosed
embodiments. In the beginning, cells are dispersed in a suspension
(202). For adherent cells, they can be lifted and dispersed into a
suspension. Cells can be washed when desired. Further, the cell
concentration can be determined by a counting method (204). An
appropriate number of cells are then taken for a monolayer or for
multiple monolayers. Further, the cell suspension is adjusted to a
suitable volume for a sample container (206). Target substances to
be delivered may be included in the cell suspension before loading
the cell suspension to sample containers. Further, the cell
suspension is loaded in a sample container (208).
[0124] After loading the cell suspension in a sample container, a
cell monolayer is arranged (210). If the cell monolayer is to be
made by gravity mediated natural sedimentation, the sample
containers need to be placed on a level surface for a certain
period of time. The time needed to form a monolayer may be
determined empirically using the methods discussed in paragraph
[0068]. For example, the formation of the monolayer may be observed
under microscope. If the cell monolayer is to be made by
centrifugation, the sample containers can be placed in a
centrifuge. After the formation of the cell monolayer, the cells
are treated by an electrical pulse (212). The electrical pulse
treatment may be performed after centrifuge or during centrifuge if
a centrifuge is used to form the cell monolayer. After the
electrical pulse treatment of the cells, the cells are unloaded
from the sample containers (214). If the electrical pulse treatment
is during the centrifuge, the centrifuge is stopped before the
unloading of the cells. For electrical cell fusion, there may be an
additional step in making the cell double-monolayer and the buffers
and the electrical pulse can be different from those typically used
for the purpose of substance delivery.
[0125] The present disclosure may also be applicable to
electroporation in cell suspension. FIG. 13A illustrates an
exemplary sample container 70 consistent with the disclosed
embodiments, which may be used for electroporation of a cell
suspension. As shown in FIG. 13A, the container 70 includes a
cylindrical insulator chamber 71, a fixed electrode 75, a movable
electrode 76 that also functions as a sealing cover, an open
insulator cover 72, and an excess-receiving groove 74.
[0126] The insulator chamber 71 has an open groove at the bottom
end for fixing the electrode 75 and another open groove 74 at the
top of the wall of the chamber 71 to receive the movable electrode
76. The diameter of the electrode 75 may be slightly larger than
the diameter of the groove at the bottom end of the insulator
chamber 71 so that the electrode 75 can be tightened by some
tension generated in the bottom end of the insulator chamber 71.
Alternatively the electrode 75 may be fixed to the insulator
chamber 71 for sealing of the container bottom by gluing or any
other appropriate methods.
[0127] The movable electrode 76 is embedded in the open insulator
cover 72 that is connected to the main insulator chamber 71 through
a flexible linkage 73. The cover 72 is capable of fitting in the
top groove 74 in the wall of the main insulator chamber 71 tightly,
allowing the electrode 76 to cover on an inner rim in the main
insulator chamber 71 and seal the sample. To securely seal the cell
suspension 77 without air bubbles, the volume of the cell
suspension to be added may be slightly larger than the allowed
volume in the sealed container so that there is a little excess
liquid to ensure a perfect sealing. The excess liquid pushed out by
closing down the electrode cover 76 can flow to the
excess-receiving groove 74 etched at the top of the wall of the
main insulator chamber 71. After electroporation, the cells in the
excess liquid can be discarded as they are not electroporated.
While it is a good practice to seal a cell suspension inside the
container 70 without air bubbles, some small air bubbles may
actually be tolerated as long as the small air bubbles only occupy
a small portion of the electrode surface.
[0128] The understandings of the longitudinal shielding effect and
the lateral enhancing effect can also help to improve the
electroporation efficiency for cells in suspensions. For cell types
of larger sizes, they require lower field strength to obtain
effective electroporations and cell heterogeneity caused by
proximal cell-to-cell electrical interactions may not be beneficial
for electroporation efficiency. However, for more common cell types
of smaller sizes that require a high field strength to obtain
effective electroporation, cell heterogeneity caused by proximal
cell-to-cell electrical interactions can instead be very beneficial
for cell electroporation.
[0129] For smaller cells, increasing the cell concentration in the
suspension may increase the effect of lateral enhancing.
Simultaneously longitudinal shielding effect may be increased as
well. The increased longitudinal shielding may be a worthy tradeoff
to the increased lateral enhancing effect. The longitudinal
shielding effect may be alleviated by an alternating-current
pulsing scheme.
[0130] The preferred percentage of total cell volume in the cell
suspension would be around or larger than 10%. When there are not
enough cells, artificial insulator particles may be used similar to
those described for the monolayer based methods.
[0131] For improved electroporation of cell suspensions, the
exemplary sample container 70, or other sample containers which are
similar to those described for monolayer based methods may be used.
The container 70 dedicated for electroporation of cell suspensions
tends to have longer distance between the electrodes 75 and 76 so
that fewer cells are within the immediate vicinity of the
electrodes to reduce electrochemical toxicities to the cells. The
distance between the electrodes 75 and 76 is preferred to be
between 3 mm to 100 mm, and a distance between 5 mm to 50 mm is
further preferred.
[0132] The container 70 may be in a shape of a cylinder. The
diameter of the cross section of the container 70 may be larger
than 1 mm. In certain embodiments, the diameter of the cross
section of the container 70 may be ranged from 1 to 20 mm. Other
diameter value may also be used. The container 70 may also be in
other shape with a comparable cross section area to that of a
cylindrical container.
[0133] The movable electrode such as the electrode 76 in the
container 70 may be used on both ends of an insulator chamber,
especially when the inner diameter of the container is small or the
distance between the electrodes is quite long. For such a container
70, it may not be convenient to load a sample with just one movable
electrode. Since the cells near the electrodes are subject to
significant electrochemical toxicity, some markings may be made on
the insulator chamber 71 as indicators of the harmed cells to be
discarded. The container 70 may also be connected to a sample
injection system to enable continuous processing of a cell
suspension sample that flows into the container.
[0134] When the container 70 has a relatively long distance between
the electrodes 75 and 76, a high voltage would be needed to
maintain a comparable electric field strength. The seeming
disadvantage to require the pulse generator to deliver a higher
voltage may be less of a problem.
[0135] For example, to electroporate a suspension of a human cell
line, 200 volts is needed for a 0.2 ml sample in a 4 mm-gap cuvette
with exponential discharge from a capacitor of about 1000 .mu.F. If
the same 0.2 ml cell suspension is placed in a longer container
with 2 cm electrode distance (5 times of cuvette gap distance), the
voltage required would be 1000 volts, but a capacitor of only 40
.mu.F ( 1/25 of 1000 .mu.F) is needed because the electric energy
from a capacitor follows the equation of
E=0.5U.sup.2C
where E is electric energy, U is voltage and C is capacitance.
Therefore a high voltage pulse generator is actually easy to
manufacture because it needs a much smaller capacitor to store a
similar amount of energy. Similarly, it would not be difficult to
generate other wave forms of higher voltages.
[0136] Very small cells such as bacteria need a very high electric
field strength such as 20,000 V/cm to be electroporated in a
low-conductance liquid such as water. Traditionally bacteria
electroporation is done in a cuvette with a short electrode
distance of 1 mm or 2 mm so that a voltage of less than 3,000 V is
usually needed. To electroporate very small cells such as bacteria
in the container 70, an ultra-high voltage may be needed.
Ultra-high voltage, as used in this disclosure, refers to the
voltage that is higher than 5,000 V and often in the range of
10,000 to 30,000 V typically used for electroporation of very small
cells. A pulse generator capable of delivering tens of thousands
volts may not be difficult to manufacture, since a very low
capacitance would be needed to store the energy under ultra-high
voltages. The pulse generators can be equipped with rechargeable
batteries to become cordless and facilitate easy mobility.
[0137] The sealable container 70 with longer electrode distance may
effectively prevent electric arcs. In a traditional cuvette,
unevenly distributed ionic solutes may form small leaky areas for
electric current that short-circuit the two electrodes nearby. In a
sealed container with a longer electrode distance, even when a
small leaky area forms, it would be far less likely to extend from
one electrode to the other to cause short-circuit. Even when there
are small air bubbles trapped near the electrodes, they would not
cause an arc because they do not extend from one electrode to the
other. Therefore, the longer sealed container 70 would be
advantageous in suppressing the formation of an electric arc.
[0138] Another advantage of the longer container 70 is that it is
easier to manufacture the container 70 in high precisions. A 50
.mu.m distance error is 5% for a 1 mm cuvette, but it is only 0.25%
for a 2 cm long container.
[0139] FIG. 13B illustrates an exemplary use of a lower medium
layer 78 in the container 70 for electroporation of cell suspension
consistent with the disclosed embodiments. As shown in FIG. 13B,
the lower medium layer 78 is formed. A cell suspension 77 is loaded
on the lower medium layer 78. The cell suspension 77 may be brought
into a pellet 79.
[0140] The lower medium layer 78 is similar to the one used for
monolayer method of electroporation, such as the lower medium layer
12 in FIG. 1A. The lower medium layer 78 physically keeps cells
away from the electrode 75. Another medium layer similar to 78 can
be used on the upper electrode as well, especially when the
container is intended for electroporation of a cell suspension, so
that both ends of the cell suspension are protected from direct
exposure to the electrodes. The cell suspension 77 may be
electroporated directly in this container, or the cells in the
suspension may be brought into the pellet 79 to increase the cell
concentration. While the container 70 for electroporation of a cell
suspension tends to have longer distance between the electrodes 75
and 76, the container 70 dedicated for electroporation of the cell
pellet 79 can have a shorter distance between the electrodes 75 and
76. The pellet 79 can be made by the natural gravity or by
centrifugation. If a centrifuge is used, pulsing of the cell pellet
79 can take place after centrifugation or during centrifugation,
similar to the methods described for monolayer and
multiple-monolayer electroporations in the centrifuge 50. The
pellet 79 can be electroporated with high efficiency and low
toxicities to the cells at a lower voltage than what is need for
electroporation of the same cells in a suspension. The pulse
generators can be equipped with rechargeable batteries to
facilitate easy mobility and the variations of the pulse generators
described for monolayer based electroporation may also be applied
for electroporation of cells in suspensions or pellets. Artificial
insulator particles may also be used with the real cells for making
cell pellets.
[0141] The present disclosure provides electroporation devices and
methods that can achieve high-efficiency and low toxicities in
electroporation. The devices and methods according to the present
disclosure offer advantages over other methods and devices. For
example, the devices and methods according to the present
disclosure have certain advantages over the capillary
electroporation.
[0142] FIG. 14 illustrates an exemplary capillary assisted
electroporation. As shown in FIG. 14, the electroporation is
carried out in a capillary with an inner diameter of 20 .mu.m. Two
cells, cell A and cell B, are located within the capillary. Cell A
has a diameter of 18 .mu.m, and cell B has a diameter of 16
.mu.m.
[0143] As shown in FIG. 14, the smallest distance between the cell
A and the capillary wall is 1 .mu.m and the smallest distance
between the cell B and the capillary wall is 2 .mu.m. The capillary
wall is an insulator so that the electric current is restricted to
the gap between the cell and the capillary wall. The same total
electric current flows through the gaps around the cell A and the
cell B.
[0144] Because the cross-sectional area of the gap around cell B is
about twice of the cross-sectional area of the gap around cell A,
the electric field strength in the gap around cell B is only about
half of the field strength in the gap around A. Therefore, the
transmembrane potential of the cell A beyond the gap area is about
twice (200%) of the transmembrane potential of the cell B, although
the diameter of A is only 1/8 (12.5%) more than the diameter of B.
In the capillary, cell A is benefited with the lateral enhancing
effect from the capillary wall. However, it is very difficult for B
to be electroporated, although B is only slightly smaller. Even
with an alternating current pulsing scheme, the cell B would have
about half of the transmembrane potential of A. As a result, cell B
still would not be effectively electroporated. If the diameter of A
becomes 19 .mu.m and its gap becomes 0.5 .mu.m, the transmembrane
potential of A would become about 400% of B, suggesting that a
capillary could introduce a significant amount of cell
heterogeneity in electroporation efficiency.
[0145] A capillary thus works by employing the lateral enhancing
effect from the capillary wall. It works better when the inner
diameter is smaller because a higher portion of the cells are
located within the immediate vicinity of the capillary wall for a
smaller capillary. The compact monolayer based electroporation
method reduces cell heterogeneity in electroporation. By
comparison, the capillary method of electroporation exaggerates the
cell heterogeneity in electroporation and it is inherently limited.
Primarily utilizing the lateral enhancing effect from the
concentrated cells themselves in a suspension or a pellet as
described in the present disclosure can be advantageous in both
electroporation efficiency and cost effectiveness.
[0146] While various embodiments and the accompanying figures have
been shown and described, it is understood that they are not
intended to limit the scope of the present invention.
* * * * *