reversible permeabilization using high-intensity femtosecond laser pulses: applications to...

Reversible Permeabilization UsingHigh-Intensity Femtosecond Laser Pulses:Applications to Biopreservation

Vikram Kohli,1 Jason P. Acker,2,3 Abdulhakem Y. Elezzabi1

1Ultrafast Photonics and Nano-Optics Laboratory, Department of Electrical andComputer Engineering, University of Alberta, Edmonton, Alberta, Canada2Canadian Blood Services, Research and Development, 8249-114 Street, 3rd Floor,Edmonton, Alberta, Canada T6G 2R8; telephone: þ780-702-8629; fax: þ780-702-2501;e-mail: [email protected] of LaboratoryMedicine and Pathology, University of Alberta, Edmonton,Alberta, Canada

Received 8 May 2005; accepted 23 June 2005

Published online 27 September 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20689

Abstract: Non-invasive manipulation of live cells isimportant for cell-based therapeutics. Herein we reporton the uniqueness of using high-intensity femtosecondlaser pulses for reversibly permeabilizing mammaliancells for biopreservation applications. When mammaliancells were suspended in a impermeable hyperosmo-tic cryoprotectant sucrose solution, femtosecond laserpulses were used to transiently permeabilize cells forcytoplasmic solute uptake. The kinetics of cells exposed to0.2, 0.3, 0.4, and 0.5 M sucrose, following permeabiliza-tion, were measured using video microscopy, and post-permeabilization survival was determined by a dualfluorescence membrane integrity assay. Using appropri-ate laserparameters,weobserved thehighest cell survivalfor 0.2 M sucrose solution (>90%), with a progressivedecline in cell survival towards higher concentrations.Using diffusion equations describing the transport ofsolutes, the intracellular osmolarity at the inner surfaceof the membrane (x ¼10 nm) and to a diffusive length ofx ¼10 mm was estimated, and a high loading efficiency(>98% for x ¼10 nm and >70% for x ¼10 mm) wascalculated for cells suspended in 0.2 M sucrose. This isthe first report of using femtosecond laser pulses forpermeabilizing cells in the presenceof cryoprotectants forbiopreservation applications. � 2005 Wiley Periodicals, Inc.

Keywords: femotsecond; ultrashort laser pulses; per-meabilization; cryoprotectant; sugars; biopreservation;cryobiology; cryopreservation

INTRODUCTION

With the recent advancements in tissue engineering, cell

transplantation, and genetic technologies, living cells as a

therapeutic tool for clinical care have receivedwide attention

(Acker et al., 2004). These emerging technologies depend on

the characteristics of living cells, and thus maintaining and

preserving the biological function of cell-based therapeutics

remains one of the most important challenges facing

reparative medicine (Acker, 2005).

The goal of biopreservation is to protect the integrity

and functionality of living cells, tissues, and organs, which

has resulted in the development of techniques that can

achieve biological stability and ensure a viable state follow-

ing ex vivo storage (Acker, 2005; Acker et al., 2004).

Cryopreservation has been the traditional approach used for

long-term preservation. In this process, cells are frozen

to ultra low temperatures (i.e., �80 to �1968C) where

molecular motion is suppressed and biochemical and meta-

bolic reactions are arrested. Achieving cell preservation at

cryogenic temperatures requires a proper balance between

the cooling rates, thawing rates, and cryoprotectant concen-

tration (Diller, 1975; Fahy et al., 1984; Lovelock, 1953;

Mazur, 1984).

Efforts to improve post-thaw cell survival have focused

on the role of cryoprotectants in stabilizing and protecting

cells from lethal injury. Low-molecular weight permeating

cryoprotectants such as methanol, ethylene glycol, glycerol,

and dimethyl sulfoxide have been routinely used in cryo-

preservation studies (Lovelock, 1953; Palasz and Mapletoft,

1996). However, more recently, low concentrations of mem-

brane impermeable disaccharides (trehalose and sucrose),

have received wide attention (Acker, 2005; Acker et al.,

2004; Chen et al., 2001; Crowe et al., 2001; Fabbri

et al., 2001). The mechanism of sugar protection is an active

area of research that includes the role of the glassy state in

long-term stabilization (Buitink et al., 1998; Crowe et al.,

1998), the interaction of sugars with biological molecules

and supramolecular structures to afford stabilization (Crowe

et al., 1988) and the role that the unique physico-chemical

properties of sugars have during freezing and thawing

(Crowe et al., 2004). Regardless of the mechanism of action,

�2005 Wiley Periodicals, Inc.

Correspondence to: J.P. Acker

Contract grant sponsors: Canadian Blood Services (CBS); Natural

Sciences and Engineering Research Council of Canada (NSERC); Infor-

matics Circle of Research Excellence (iCORE); Canada Foundation for

Innovation (CFI)

the protective effect of cryoprotective sugars has been

demonstrated in a wide variety of biological systems (Acker

et al., 2003; Buchanan et al., 2004; Chen et al., 2001;

Crowe et al., 2001; Eroglu et al., 2000, 2002; Satpathy et al.,

2004).

Since non-reducing disaccharides are impermeable to the

lipid bilayer, several methods have been used for permeabi-

lizing cells for cytoplasmic sugar uptake. These include

microinjection, osmotic and thermal shock, genetic engi-

neering, electroporation, and the use of bacterial pore-

forming toxins (Acker et al., 2003; Buchanan et al., 2004;

Eroglu et al., 2000, 2002).While each method has significant

benefits, the tedious process of microinjection (Acker et al.,

2004) and the irreversible cell damage induced by electro-

poration (including membrane bleb formation, disruption of

biochemical pathways, DNA denaturation, and cell lysis

(Acker et al., 2004; Kinosita and Tsong, 1977; Tsong, 1991;

Vernhes et al., 1999; Weaver, 1993) have limited their use in

clinical applications. Genetic engineering and thermal and

osmotic shock have been shown to be effective at overcoming

the impermeability ofmammalianmembranes, but the extent

to which sugars are accumulated intracellularly may be

insufficient to confer protection (Acker et al., 2004).

Furthermore, bacterial a-toxins induce irreversible mem-

brane damage and may have potential cytotoxic and

immunogenic effects (Acker et al., 2003; Buitink et al.,

1998; Tsong, 1991; Thelestam, 1983). In order to achieve

high post-permeabilization and post-thaw survival rates, it is

important that the permeabilization tool be applicable to all

cell types without inducing deleterious effects.

Recently, the application of high-intensity ultrashort

(femtosecond) laser pulses has been shown to have important

implications for studying live cells (Koenig et al., 1999;

Kohli et al., 2005; Matsunaga et al., 2004). The lasers

contact-free and non-invasive nature allows for cells to be

manipulated in a precise and controlled fashion. Since

the pulse duration of femtosecond lasers is shorter than

the thermal diffusion time (picoseconds to nanoseconds),

thermal shock and mechanical damage is insignificant

(Kohli et al., 2005; Tirlapur and Koenig, 2002a,b; Niemz,

2002).

The purpose of this study was to evaluate the effective-

ness of using femtosecond laser pulses to reversibly

permeabilize mammalian cells. Our goal was to demon-

strate the generation of transient optical pores, and the

cytoplasmic uptake of cryoprotectant sugar. We evaluated

the kinetics of cells suspended in a hyperosmotic solution of

sucrose, and measured the volumetric change upon per-

meabilization. Using appropriate laser parameters, the

optimal cell survival rate as a function of cryoprotectant

concentration was determined. To ensure efficient post-

permeabilization cryoprotectant loading, the solute trans-

port equation for a porous membrane was used to estimate

the intracellular accumulation of sugar. To date, no report

has documented the applicability of using ultrashort laser

pulses for intracellular delivery of sugar for biopreservation

applications.

MATERIALS AND METHODS

Cell Culture

Madin-Darby Canine Kidney cells (MDCK; American Type

Culture Collection (ATCC) CCL-34) were cultured at 378Cin an atmosphere of 95% air plus 5% carbon dioxide in

supplemented medium consisting of minimum essential

media with Hanks salts, 16 mmol/L sodium bicarbonate,

2 mmol/L L-glutamine, and 10% fetal bovine serum (all

components from Hyclone Laboratories, Logan, UT). Cells

in exponential growth phase were harvested by exposure to a

0.25% trypsin–1 mM EDTA solution at 378C, washed twicewith supplemented medium, plated onto sterile, micropat-

terned glass coverslips (12mm2FisherBrand) and cultured at

378C for 12 h to allow the cells to attach.

Cell Micropatterning

A simplified cell micropatterning method (Chen et al., 1998)

using a polydimethylsiloxane (PDMS) elastomeric stamp to

create non-adhesive regions of agarose on glass substrate

allowed for precise control of cell geometry. Using micro-

patterned cells allowed for accurate retrospective analysis of

cell viability following laser permeabilization.

Photolithography

Four-inch silicon wafers were bathed in a piranha solution

consisting of sulphuric acid (J.T. Baker, Phillipsburg, NJ) and

hydrogen peroxide (J.T. Baker) in a 3-to-1 ratio for 15 min.

The wafers were rinsed in deionized water and dried using

nitrogen. A layer of hexamethyldisilazane (Arch Chemicals,

Norwalk, CT) was spin-coated onto the silicon wafers and

baked at 1508C for 10 min to improve photoresist adhesion.

A 10 mm layer of Microposit SJR 5740 positive photoresist

(Shipley,Marlborough,MA)was spin-coated onto thewafers

and baked at 1158C for 6min. For pattern transfer, the wafers

were exposed for 18 s to ultraviolet light at a constant

intensity of 18.1 mW/cm2. The pattern was transferred from

an acetate mask produced by high-resolution printing

(Advance Reproductions, North Andover, MA) and con-

sisted of a matrix of rectangles of dimensions 30� 30 mmwith a well separation of 60 mm.

Preparation of the PDMS Stamps

PDMS stamps were prepared by mixing prepolymer and a

curing agent in a 10:1 ratio by weight as indicated by the

manufacturer (Corning, NY). The polymerwas cured at 708Cfor 1 h in a vacuum, and was peeled off the wafers and used

for patterning of glass substrates.

Patterning of the Glass Coverslips With Agarose

PDMS stamps were placed pattern side down on glass

coverslips. A 0.6 g/100 mL low-melting point agarose

890 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 92, NO. 7, DECEMBER 30, 2005

(Invitrogen Corporation, Carlsbad, CA)/40% ethanol (VWR

Can Lab, Brampton, Ont., Canada) solution was prepared

and injected through the channels of the micropatterns. The

coverslips were placed in a vacuum chamber for 18 h to dry.

Following drying, the stamps were peeled off, leaving

patterned agarose on the glass substrate.

Cell Viability Assessment

A dual fluorescent staining technique was used for quanti-

tative assessment of the integrity of the cell plasma

membrane (Yang et al., 1998). SYTO 13TM (Molecular

Probes, Eugene, OR) and ethidium bromide (EB; Sigma

Chemical Company, Mississauga, Ont., Canada) were used

to differentially stain the cells. SYTO readily permeates

intact cells and fluorescently labels both RNA and DNA

green. EB has been shown to penetrate only cells with

damaged membranes and forms a fluorescent red complex

with nuclear DNA (Edidin, 1970). The SYTO/EB assay for

membrane integrity has been previously shown to correlate

well with other assessment techniques for cell viability (Yang

et al., 1998). Percent survival based on membrane integrity

was calculated as the number of SYTO positive cells over the

total number of cells.

Cell Volume Measurements

The volumetric response of micropatterned MDCK cells in a

hypertonic solution of sucrose was used to assess solvent

mass transport. The estimated volumetric response of the

cells to a transient pore was determined by measuring the

diameter of the cell, along both the x- and y-axis, as a function

of time using image analysis software (ImageJ, National

Institutes of Health). The measurements were averaged

to minimize errors in the approximation of the volume.

Assuming a spherical geometry, cell volumes were then

calculated.

Intracellular Solute Delivery Estimation

A modified diffusion equation for solute transport through a

porous membrane was used to estimate the intracellular

accumulation of cryoprotectant sugar for cells in 0.2, 0.3, and

0.5 M sucrose suspensions. From Fick’s law of diffusion, the

solute flux rate of sucrose in water is (Finkelstein, 1987)

Fsw ¼ Dsw

DCs

Dxð1Þ

where Fsw is the flux transport rate of the solutes in water,

Dsw is the diffusion coefficient of the solutes in water, DCs is

the solute concentration difference across the cell mem-

brane, and Dx is the distance defined by DCs. Since sucrose

is impermeable to the plasma membrane, the permeabiliza-

tion of the cell by femtosecond laser pulses provides the

transport mechanism for cytoplasmic sugar uptake. Equa-

tion (1) can then be modified to represent the solute

transport flux through a pore (Finkelstein, 1987)

Fsw ¼ nA

LDsw

DCs

Dxð2Þ

where A is the area of the pore, L is the length of the pore,

and n is the number of pores. Absorbing A, L, n, Dx, and Dsw

into a constant, denoted as D0, noting from the continuity

equation that the one-dimensional time rate of change in

solute concentration is (Smits, 2000)

@Csðx; tÞ@t

¼ *r � Fsw ð3Þ

and substituting Equation (2) into Equation (3), then solving

for Cs (x, t) subject to initial conditions, Cs (0, t)¼C0 and Cs

(1, t)¼ 0

Csðx; tÞ ¼ C0erfcx

2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðnDswAt=LDxÞ

p !

ð4Þ

yields a modified equation for Fick’s 2nd law of diffusion

describing solute flux through a membrane pore. In

Equation [4] Cs (x, t) is the concentration of solutes, C0 is

the initial concentration of solutes, t is time, x is the distance

over which Cs (x, t) is computed, and erfc is the

complementary error function. The length of the pore is

a constant equal to the thickness of the cell membrane

(L¼ 10 nm), and A was taken to be the area of the pore

generated by the focused femtosecond laser pulses, where

the diameter was equal to the laser spot size. Cs (x, t) was

calculated as a function of distance along the pore, with C0

equal to the extracellular osmolarity, and t was set to the

time required for the cell to reach equilibrium volume post-

permeabilization. The diffusion coefficient of sucrose in

water was determined using Einstein’s fluctuation–dissipa-

tion formula, where a single sucrose molecule was modeled

as a sphere with a defined diameter. To estimate the diameter

of a sucrose molecule, bond lengths were added along the

long axis yielding a calculated diameter of 1.6 nm.

Laser Microscope Setup

Optical permeabilization ofmicropatternedMDCKcells was

achieved using a modelocked titanium sapphire laser

oscillator, producing sub-10 femtosecond laser pulses, with

a center wavelength of 800 nm and a repetition rate of

80 MHz. The laser pulses were coupled to a modified optical

microscope and directed towards the biological sample, as

shown in Figure 1. To focus the femtosecond pulses, a 0.95

high numerical aperture microscope objective was used,

producing a spot size of <1 mm. Using an average delivered

power of 270–410mW, 3–5 nJ pulses were generated within

the focal volume and dynamically gated using a mechanical

shutter.MicropatternedMDCKcells were placed on an x–y–

z translation stage for precise sample movement, and the

stage was temperature controlled to 48C. Optical permeabi-

lization was viewed with a charge coupled device mounted

on the modified optical microscope and captured using

commercial video software.

KOHLI ET AL.: REVERSIBLE PERMEABILIZATION USING FEMTOSECOND LASERS 891

Statistical Analysis

Unless otherwise stated, all uncertainty calculations were

determined using the error propagation formula. Assuming

that fluctuations in the function were uncorrelated, all

covariant terms were set to zero.

RESULTS

Laser Induced Optical Poration

Before permeabilizing mammalian cells in the presence of

impermeable cryoprotectants, we first assessed the feasi-

bility of using a femtosecond laser to create optical pores

in micropatterned cells. Using an average laser power of

410 mWand a gated shutter time of<10 ms, sub-10 fs laser

pulses were coupled and focused by a 0.95 high numerical

aperture microscope objective. A focal spot of <1 mm in

diameter was generated at the focal plane of the membrane,

with a total energy absorption of 4 mJ over the gated

femtosecond pulse train. To evaluate the presence of a pore,

the dual fluorescence membrane integrity assay SYTO/EB

was used. The mammalian cells were stained �10–20 min

post-laser exposure. Figure 2a depicts bright field images

of micropatterned cells before laser exposure, where the

arrows indicate cells that have been selectively targeted for

permeabilization. In Figure 2b, a fluorescence image after

permeabilization is shown. Micropatterned MDCK cells

exposed to femtosecond laser pulses were positive for

EB staining, indicating a damaged permeable plasma

membrane.

Intracellular Delivery ofSucrose Using Optical Pores

To determine whether optical pores are sufficiently large

and open for a duration that permits sucrose diffusion, the

volumetric response of MDCK cells suspended in 1.0 M

sucrose was evaluated. Figure 3a depicts the shrunken

appearance of three MDCK cells suspended in a hypertonic

solution of 1.0M sucrose. In Figure 3a, a single cell has been

selected for permeablization, and the focused femtosecond

laser spot is shown. When the ultrashort (femtosecond)

pulses were focused to a spot size of <1 mm, a transient

optical pore was created in the individual cell, exposing the

cytoplasm to the extracellular environment. Once exposed,

Figure 1. Experimental setup. A Kerr lens modelocked titanium sapphire laser oscillator, producing sub-10 femtosecond laser pulses, with a center

wavelength of 800 nm and a repetition rate of 80MHzwas directed towards a 0.95 high numerical aperturemicroscope objective. The ultrashort (femtosecond)

pulses were focused onto the cell with a focal spot of <1 mm. The cells were placed on a temperature-controlled stage cooled to 48C. To visualize reversiblepermeabilization, the cells were illuminated from beneath with white light, and imaged using a charge coupled device (CCD) and processed by commercial

video capture software.


an increase in cellular volume was observed, Figure 3b, with

the increase occurring only in the targeted cell. To deter-

mine the hypertonic to equilibrium volumetric response of

micropatterned MDCK cells, the volume was calculated

(presuming a spherically shaped cell) before and after

permeabilization. In hypertonic conditions, an equilibrium

V/Vequil¼ 0.578� 0.085 was measured versus V/Vequil¼1.000� 0.055 at equilibrium volume post-permeabilization.

Therefore, an approximate 60% change in volume was

observed when the cell was porated using femtosecond laser

pulses in the presence of 1.0 M sucrose.

The kinetics of MDCK cells suspended in various

hyperosmotic solutions of sucrose were also measured.

Using video microscopy, the change in volume of cells

porated with femtosecond laser pulses was measured as a

function of time. Before permeabilization, the initial equili-

brium cell volume in the solution was determined, and upon

poration, the return to equilibrium volume was measured.

Figure 4 depicts the kinetics of a single mammalian cell

suspended in a 0.2 M sucrose solution. All calculated values

for the volume were normalized to the post-permeabilized

equilibrium volume, as determined from direct measure-

ments. Similar volumetric response graphswere observed for

micropatterned MDCK cells suspended in 0.3 and 0.5 M

sucrose (data not shown). Cells suspended in 0.2 M sucrose

took less than 270 ms to return to equilibrium volume

following laser permeabilization.

Figures 5 and 6 illustrate the results of our laser perme-

abilization method for MDCK cells in a 0.2, 0.3, 0.4, and

0.5 M sucrose solution. The bright field image in Figure 5

represents live cells suspended in a 0.2 M hyperosmotic

sucrose solution pre-laser exposure, while the fluorescence

images depict cell viability 30–40 min post-exposure.

In Figure 5, a total of six cells were laser exposed (cell 5

detached from the well post-permeabilization), indicated by

the numbers 1–6 (fluorescence and bright field images for

0.3, 0.4, and 0.5 M not shown). The fluorescence images

in Figure 5 depict live MDCK cells, which have intact

membranes and optical pores that have completely sealed.

This is evident from the absence of EB diffusion, which

stains the cells red-orange for membranes that have been

compromised.

Figure 2. Laser-induced optical pore. a: Represents a bright-field image of live micropatternedMDCK cells. Using an average laser power of 410mW, and a

gated shutter time of<10ms, sub-10 fs laser pulses were coupled and focused by a 0.95 high numerical aperturemicroscope objective to generate a sub-micron

focal spot. The arrows in (a) represent cells selected for laser permeabilization. Approximately 10–20 min post-permeabilization the cells were stained with

SYTO/EB to evaluate the presence of a pore. b: The fluorescent image depicts the cytoplasmic uptake of EB for laser-permeabilized cells, as represented by the

arrows. Scale bar in (a) is 40 mm; scale bar in (b) is 100 mm. [Color figure can be seen in the online version of this article, available at www.

interscience.wiley.com.]

Figure 3. The response of a micropatternedMDCK cell suspended in 1.0 M sucrose when permeabilized by femtosecond laser pulses. a: MDCK cell before

permeabilization. The arrow depicts the focused femtosecond laser spot. Only one cell was chosen for permeabilization, demonstrating the precision of the

process. b:MDCK cell after permeabilization. The cell has increased in cellular size towards equilibrium volume. The arrow in (b) illustrates the permeabilized

cell. Scale bar in (a) and (b) is 40 mm.


When the micropatterned MDCK cells were suspen-

ded in an increasingly hypertonic solution (0.3 M; n¼ 7,

0.4 M; n¼ 15, 0.5 M; n¼ 8), post-permeabilized cells

exhibited an increase in membrane damage. Figure 6

illustrates cell survival as a function of sucrose concentra-

tion under constant laser power and energy absorption.

A post-laser exposure survival rate of 91.5� 8% was

achieved using an extracellular sucrose concentration of

0.2 M.

Percentage Intracellular Uptake

Using Equations (2–4), an estimate of the intracellular solute

delivery was calculated, and is presented in Table I. From

Equation (4), with n¼ 1, the solute concentration, Cs (x, t),

was computed for each cryoprotectant sucrose solution,

where t for each solutionwas determined from thevolumetric

response plots at equilibrium volume post-permeabilization.

Figure 7 depicts the change in solute concentration as a

Figure 4. Volumetric response of a micropatternedMDCK cell in a 0.2 M

cryoprotectant sucrose solution. Initially the cell is in a shrunken state.

Upon laser permeabilization, the cell quickly swells to equilibrium volume.

The value ofVequil was taken to be the equilibrium volume asmeasured using

ImageJ.

Figure 5. Laser permeabilized micropatterned MDCK cells suspended in a 0.2 M cryoprotectant sucrose solution. a: Represents bright field images of

micropatterned cells where the numbers indicate cells selected for permeabilization. All cells were treatedwith an average laser power of 270mWusing a gated

shutter time of<10 ms with a total energy absorption of<2 mJ. After treatment, a change in cell volume from hypertonic equilibrium volume to equilibrium

volumewas observed. b: The cells were stained with SYTO/EB approximately 30–40 min post-laser exposure. The absence of EB diffusion indicates that the

cells were permeabilizedwith reversible pores. Cell 5 detached from themicropatternedwell post-permeabilization. Scale bar in (a) is 40 mm; scale bar in (b) is

100 mm. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

Figure 6. Cell survival post-laser exposure as a function of the

extracellular solute concentration. A progressive decline in cell survival

with increasing solute concentration is observed. Highest cell viability was

obtained forMDCKcells suspended in 0.2Msucrose.No cells survived post-

laser exposure in a 0.5 M sucrose suspension.


function of distance through the pore for cells in a 0.2 M

sucrose suspension. Since the average thickness of a cell

membrane is 10 nm, diffusion of solutes to a distance of

x¼ 10 nm would constitute intracellular sucrose accumula-

tion. Cs (10 nm, tequil) from Figure 7 for cells suspended

in 0.2 M sucrose was estimated to be Cs (10 nm, tequil)¼0.199 M with a loading efficiency of 99% to a distance just

inside the plasma membrane. Similar diffusion profiles were

obtained for 0.3 and 0.5 M sucrose. Cs (10 nm, tequil) for

0.3 and 0.5 Mwere estimated to be 0.299 and 0.499Mwith a

loading efficiency of 99% for each osmolarity. The inset in

Figure 7 represents the diffusion profile plotted to large

diffusion lengths. From the inset, the sucrose concentration at

a distance of x¼ 10 mm within the cell was estimated to be

Cs (10 mm, tequil)¼ 0.145, 0.198, and 0.330 M for cells

suspended in 0.2, 0.3, and 0.5 M sucrose.

DISCUSSION

Induced Transient Pores byFemtosecond Laser Pulses

Intracellular sugars are increasingly being used in the

development of effective technologies for the preservation

of cells and tissues used in reparative medicine. To assess the

usefulness of using femtosecond laser pulses for biopreser-

vation applications, we first evaluated their ability to create

transient pores for the selective uptake of cryoprotectants. As

shown in Figure 2, the creation of optical pores allows for

intracellular solute uptake.When the cells were suspended in

SYTO/EB, the localized pore facilitated the diffusion of EB

into the cytoplasm of the cell. Since the molecular weight of

EB (394 g/mol) and sucrose (342 g/mol) are comparable, the

diffusion of EB suggests that the optical pore should be large

enough to allow the cytoplasmic uptake of cryoprotective

disaccharides. In Figure 2b, the red labeled complex stains

for membrane integrity, indicating that the porated cells are

compromised. Proper adjustments to pulse energy, pulse

duration, and energy absorption can precisely control both

the size and dynamics of the pore. This makes femtosecond

lasers an ideal tool for microscopic manipulation of unfixed

specimens.

Determination of the Optimal Laser Parameters

We observed that focused femtosecond laser pulses could

efficiently permeabilize living cells (Fig. 2). We have

determined through previous studies (unpublished data) that

as the energy absorption of the cell increases, necrotic

behavior also increases. In order to avoid irreparable cell

damage, gating the laser pulses with a mechanical shutter of

<10 ms in duration, a total laser energy absorption of<2 mJ

provides the optimal laser parameters for optical poration

while maintaining maximized post-permeabilization survi-

val rates. Cell survival was determined by SYTO/EB, and

while the dual assay is not an absolute measure of viability, it

has beenwidely used in characterizing cell death (Chen et al.,

2001). Therefore, for all subsequent experiments we used

SYTO/EB for measuring viability, and an average laser

power of 270 mW gated at <10 ms was chosen.

Table I. Calculated values for the intracellular accumulation of sucrose at a length of 10 nm and 10 mmwithin

the cell.

Constants

Initial concentration

(molecules/cm3) tequil (s) Cs (x, tequil) (M)

A¼ 0.785� 0.392 mm2 C0¼ 1.204E20 (0.2 M) tequil¼ 0.20 (0.2 M) x¼ 10 nm

T¼ 277 K C0¼ 1.806E20 (0.3 M) tequil¼ 0.13 (0.3 M) Cs¼ 0.199 (0.2 M)

L¼ 10 nm C0¼ 3.011E20 (0.5 M) tequil< 0.13 (0.5 M) Cs¼ 0.299 (0.3 M)

Dsw¼ 2.536E-6 cm2/s Chosen to be 0.13 Cs¼ 0.499 (0.5 M)

x¼ 10 mmCs¼ 0.145 (0.2 M)

Cs¼ 0.198 (0.3 M)

Cs¼ 0.330 (0.5 M)

Figure 7. Diffusion profile for a cell suspended in an extracellular

osmolarity of 0.2 M sucrose following permeabilizaton. Using the modified

diffusion equation for a porousmembrane, the diffusion of sucrose across the

porewas calculated as a function of pore length,with t equal to the time taken

for the cell to reach equilibrium volume. For a cell membrane thickness of

10 nm, the intracellular accumulation of sucrosewas estimated to be 0.199M

at the inner surface of the plasma membrane. The inset depicts the diffusion

profile for large diffusion lengths. The concentration at a length of 10 mmwithin the cell was estimated to be 0.145, 0.198, and 0.330M for cells in 0.2,

0.3, and 0.5 M sucrose suspensions.


The Kinetics of Optical Pores

Despite the similar molecular weights of EB and sucrose, we

measured the volumetric response of mammalian cells

suspended in varying hyperosmotic solutions of sucrose as

a means to more directly monitor pore dynamics and sugar

uptake. If the transient pore provides the mechanism for

sucrose diffusion, then the kinetics should reveal a cell that

has increased in cellular volume. This method for verifying

the uptake of sucrose, based on the volumetric response, has

been employed and well characterized by Eroglu and Russo

et al. (Eroglu et al., 2000; Russo et al., 1997). Russo showed

that the volumetric response of fibroblast cells permeabilized

by a bacterial a-toxin in the presence of a hypertonic sucrosesolution was an increase in cell size towards equilibrium

volume, resulting from the intracellular uptake of sucrose

(Russo et al., 1997). Figure 4 depicts a cellular increase,

where the volume change as a function of time for a cell

suspended in 0.2 M sucrose has been plotted. Upon poration,

the cell quickly swells from hypertonic volume to equili-

brium volume. Similar volumetric plots were found for

mammalian cells in 0.3 and 0.5 M sucrose (data not shown).

The upward trend depicted in Figure 4, towards an increased

cell volume, agrees well with the volumetric response plots

found by Russo et al., and is consistent with the observed

change in cell volume due to intracellular sugar uptake

(Russo et al., 1997).

Viability of Micropatterned MDCK CellsPermeabilized by Femtosecond Laser Pulses

When live mammalian cells were exposed to high-intensity

femtosecond laser pulses, the laser was shown to be an

efficient tool for the introduction of cryoprotectants. Figure 6

depicts the survival rate as a function of increasing molar

concentrations of sucrose. As the trend suggests, cell survival

is maximized at 0.2 M (91.5� 8%) and progressively

decreases with higher sucrose concentrations. These results

are consistent with pre-freeze mammalian cells porated

using a-hemolysin by Eroglu et al., where the survival of

permeabilized fibroblast cells decreased from 98.1� 6.4 to

84.1� 3.3% using 0.2 and 0.4 M trehalose (Eroglu et al.,

2000). We found that with 0.5 M sucrose, a 0% survival rate

was observed, while a 45% increase in survival occurred

when themolar concentration was changed from 0.4 to 0.3M

sucrose. Using a 0.3 and 0.4M sucrose solution, we observed

a pre-freeze survival of 75� 5 and 30� 10%. We hypothe-

size that the progressive decline in cell survival with

increasingly hyperosmotic solutions may be due to transient

pore widening (Muldrew, 2003) leading to osmotic poration

injury (Muldrew and McGann, 1994).

Osmotic Poration Injury

Muldrew and McGann (1994) proposed an osmotic rupture

hypothesis, in which osmotically driven water induced

osmotic stress and caused rupturing of the plasmamembrane.

The stress resulted from the sudden movement of water in

response to an increasing concentration of extracellular

solutes when mammalian cells were cooled to sub-zero

temperatures. A mathematical model was developed, where

the frictional force on the membrane was converted to a

pressure term describing the pressure exerted on the

membrane resulting from water efflux (Muldrew and

McGann, 1994). Muldrew and McGann showed that the

frictional force was proportional to the water flux, Fw, and

that the pressure induced on themembrane increasedwithFw

(Muldrew andMcGann, 1994). The uniform pressure exerted

on the membrane caused a rupture, leading to a spontaneous

breakdown in the symmetry of the membrane’s tensile

strength (Muldrew and McGann, 1994).

When cells are placed in a solution containing a high

concentration of impermeant solutes, water moves out of the

cell along its concentration gradient towards a lower

chemical potential. Subsequently, the cell dehydrates until

the intracellular and extracellular osmolalities are equal.

Varying the extracellular osmolarity from a lower concentra-

tion to a higher concentration results in an increase in the

water flux across the cell membrane, as larger volumes of

water are required to dilute higher impermeant solute

concentrations. When the cells are permeabilized, water

quickly diffuses into the cell, driven by the changing solute

gradient. The passive diffusion of the solutes towards a lower

chemical potential drags the water molecules towards the

inside of the cell, and a new equilibrium cell volume is

obtained.We believe, by invoking theMuldrew andMcGann

hypothesis, that the decrease in cell survival with increasing

solute concentration can be explained by pore widening that

occurs when the trans-membrane osmotic gradient increases.

In our experiment, when femtosecond pulses were focused

onto the cell membrane, a pore of <1 mm in diameter was

created. The pore provided the transport mechanism for the

intracellular accumulation of sucrose as well as water. Since

Muldrew andMcGann showed that frictional forces arise due

to water movement across the cell membrane, we expected

similar forces to develop along the length of the transient

pore. Relating the force to pressure, the osmotically active

water diffusing through the pore would place pressure on the

pore, resulting in its widening. The amount of induced

pressure would depend on Fw, and Fw would increase with

extracellular osmolarity. We expected the pressure to be

increased by our permeabilization process, as the predomi-

nant mode of water transport would be through the pore (the

least resistive path to water movement). The uniform

pressure would be confined to the area of the pore, and not

distributed over the entire membrane, as in the case of

Muldrew and McGann (1994). The typical transit time for

water influx or efflux through a cell membrane is seconds to

minutes, where the rate of water movement is governed by

the membrane hydraulic conductivity, Lp, and the osmotic

water permeability, Pf (Elmoazzen, 2000). From Figure 4,

we notice that a cell in 0.2 M sucrose suspension reached

equilibrium volume in less than 270 ms at a temperature of

48C. Passive diffusion of water through the membrane,


facilitated by an osmotic gradient, cannot account for

the observed cellular volume increase within the defined

temperature and time. For the volume to increase towards

equilibrium in less than 270 ms would require water to be

predominately transported through the transient pore. Since

the uniformpressurewould be confined to the area of the pore,

we would expect pore widening to occur. The degree of

wideningwould increasewith extracellular osmolarity due to

the dependency of Fw on concentration. The effect of

an increasing pore size could compromise both ionic and

osmotic homeostasis, leading to cell necrosis, which may

explain the results obtained in Figure 6. A static pore sizewas

assumed in all calculations to simplify the analysis without

determining the kinetics of the pore, while giving reasonable

estimates of the intracellular concentration. Further experi-

ments examining the effect of intra- and extracellular solute

gradient on pore size and the kinetics of pore formation and

closure is required.

Pore widening has also been observed in electroporation

studies (Kinosita and Tsong, 1977; Tsong, 1991; Weaver,

1993), where membrane potentials exceeding the dielectric

strength of the bilayer resulted in pore expansion. In this

process, the pore expansion was irreversible, since the

resealing time occurred within seconds to minutes (Tsong,

1991) after the pulsewas turned off. This led to compromised

cells, subsequent to the creation of an increased pore size.

Concentration induced stress resulting from the passage of

water through a pore may contribute to decreased cell

survival with increasing extracellular osmolarity. Since the

volume of water inside the cell increases with time, a post-

permeabilized cell could swell to a critical volume, resulting

in cell rupture (Meryman, 1968, 1971). However, the contri-

bution of concentration induced stress at 0.2, 0.3, 0.4, and

0.5 M sucrose is insignificant, as Eroglu et al. reports a pre-

freeze cell survival of 84.1� 3.0% for mammalian cells

porated using a-hemolysin in the presence of 0.4M trehalose

(Eroglu et al., 2000). This is in comparison to a 30� 10%

survival using our permeabilization method, indicating that

an additional competing factor, namely the osmotic rupture

hypothesis (Muldrew and McGann, 1994), is likely respon-

sible for the large deviation in cell survival.

Theoretical Estimates of Intracellular Delivery

Equation (4) was used to estimate the percentage delivery of

intracellular sucrose at equilibrium volume. Figure 7 is a plot

of the solute diffusion profile as a function of distance along

the length of the pore for a cell in 0.2 M sucrose suspension

where tequil¼ 0.2 s. For a cell membrane thickness of L¼10 nm, the accumulation of sucrose was estimated to be

0.199 M. The concentration of 0.199 M is the osmolarity at

the inner surface of the plasma membrane, and does not

represent the final equilibrium concentration at large diffus-

ion lengthswithin the cell. The accumulation of sucrose at the

inner surface of the membrane would diffuse inside the cell,

reducing the overall cytoplasmic sugar concentration. The

inset in Figure 7 is a plot of the sucrose concentration at large

distances within the cell. At x¼ 10 mm, a concentration of

0.145Mwas estimated for cells in 0.2M sucrose suspension,

yielding a loading efficiency of 72.3%. Similar diffusion

plots for cells in 0.3 and 0.5 M sucrose were obtained, with

intracellular sucrose concentrations of 0.198 (0.3 M; 66.0%

loading) and 0.330 M (0.5M; 66% loading). A length of

10 mm was chosen based on the average radius of an MDCK

cell at isotonic volume (presuming a spherical shaped cell).

The amount of intracellular sugar can be controlled based

on the appropriate choice of laser parameters. Using the

modified diffusion equation for a porous membrane, the

solute diffusion constant was assumed to be independent

of the solute concentration. From the continuity equation,

the spatial mass transport rate was converted to a varying

concentration, where Cs (x, t) was solved using initial

conditions (Cs (0, t)¼C0 and Cs (1, t)¼ 0). If we assume

that the diffusion coefficient is not independent of the solute

concentration but linearly proportional to Cs, then D (x,

t)¼DswCs (x, t), where Dsw is the diffusion coefficient of

sucrose inwater,Cs (x, t) is the solute concentration, andD (x,

t) is the modified diffusion constant with linear dependence

on Cs (x, t) (Janavicius and Poskus, 2005). Janavicius and

Poskus (2005) solved the non-linear diffusion equation (for a

non-porous membrane) for a diffusion coefficient directly

proportional to the concentration, and found that the non-

linear equation had a diffusion profile that was smaller in

curvature than the linear diffusion equation. Essentially, the

non-linear equation resembled a linear function, and the

solution to the equation became equal to zero at a certain

distance from the diffusive source (Janavicius and Poskus,

2005). This is in contrast to the linear equation, which

asymptotically approaches zero. Despite these differences,

Janavicius showed that the predicted concentration using the

non-linear equation did not exceed the value predicted by the

linear equation by more than 20% (Janavicius and Poskus,

2005). That is, Cs (x, t) for the non-linear equation was

calculated to be higher than the prediction given by the linear

equation.

A more rigorous definition of Equation (4) would involve

a time component for both the area and length of the pore,

with proper adjustments to the diffusion constant to include

the properties of the cytoplasm. The pore generated by

the focused femtosecond laser pulses is dynamic,with its size

increasing or decreasing with time. This dynamic pore is

different from the static pore created by a-hemolysin and

other bacterial pores (Acker et al., 2003; Eroglu et al., 2000;

Russo et al., 1997). The temporal change in the pore area is

further complicated when described in conjunction with the

osmotic rupture hypothesis, since the area passes through a

maximumand changes in size due to pressure,water flux, and

membrane composition. In our analysis of intracellular

sucrose delivery, we have considered both the area and length

of the pore to be independent of time, without unreasonable

estimates in the percentage delivery (Table I). From Figure 4,

the time taken to reach equilibrium volumewas tequil¼ 0.2 s,

and this led us to conclude that the pore was open for a

duration long enough for the cell to reach this new


equilibrium volume. Consequently, the movement of water

was not limited by the time dependence of the pore area and

length. Since the average diameter of a sucrose moleculewas

calculated to be 1.6 nm, with an initial pore diameter of 1 mm,

this alsomeant that the solute fluxwas not limited by the pore

dynamics. On average, based on the ratio of the diameter of

the pore to that of the sucrose molecule, approximately 625

sucrose molecules were calculated to fit across the diameter

of the pore. In relation to Equation (4), Table I, and Figure 7, it

is not surprising to seewhy>98%of the cryoprotectant sugar

diffuses into the cell within less than 270 ms for a distance of

x¼ 10 nm and >70% to a distance of x¼ 10 mm. To further

refine this result, Einstein’s equation for the square of the

average motion of a diffusing particle can be used to estimate

the time required for a single sucrose molecule to diffuse a

predetermined distance x. Choosing the distance to be 10 nm

with Dsw equal to the value given in Table I, t¼ 0.07 ms. So,on average, a single sucrose molecule diffuses to a length of

10 nm in 0.07 ms. If the kinetics of the porewere quicker thanthe mass transport of water, then the calculated value for Cs

(x, t) at the inner surface of the membrane would provide

a concentration value much less than the extracellular

osmolarity. With the average transit time of a sucrose

molecule across the cell membrane being 0.07 ms, the rapidaccumulation of sucrose (>98% for x¼ 10 nm) within

270 ms is expected. Since the intracellular accumulation of

sugar is followed by the movement of water, it is safe to

conclude that both the flux of water and solutes towards

equilibrium volumemust have occurred before the closure of

the pore.

The unique feature of using femtosecond laser pulses

for permeabilizing cells is that the permeation rate for

cytoplasmic uptake of cryoprotectant sugar occurs at a faster

rate than that achieved by other reversible permeabilization

techniques. From Table I, we notice that for each extra-

cellular osmolarity>98% (x¼ 10 nm;>60% for x¼ 10 mm)

sucrose is delivered into the cell within milliseconds. This is

in contrast to 10–15 min required using the a-hemolysin

method (Eroglu et al., 2000), increasing the cell’s exposure

time to the permeabilizing agent.

Maximized Survival Using 0.2 M SucroseResulting From Reversible Optical Pores

Clearly, permeabilization by femtosecond laser pulses has

important consequences for biopreservation applications.

In this study, we have shown that for cryopreservation an

extracellular sucrose concentration of 0.2 M yields the

highest post-permeabilization survival rate. This is not

surprising, as other studies have reported the highest cell

survival using 0.2 M trehalose (Buchanan et al., 2004; Chen

et al., 2001; Eroglu et al., 2000, 2002). The effectiveness of

trehalose and sucrose as cryoprotectants are equal under ideal

conditions (Crowe et al., 2001), and the correlation between

sucrose concentration and cell survival is similar to that

found when using trehalose. Therefore, the choice of using

sucrose or trehalose is completely arbitrary, as both have

been shown to produce similar survival rates (Crowe et al.,

2001).

The Advantages of Femtosecond Lasers

Another benefit of femtosecond laser pulses is that they can

be adapted for precise sub-micron optical perforations

without inducing thermal pressure or shock to the biological

sample. Absorption of high intensity ultrafast laser pulses by

non-linear multiphoton absorption and ionization leads to

multiphoton electronic excitation (Noack and Vogel, 1999),

whereby energy is transported to the liberated electrons

without thermal diffusion to adjacent cellular material

(Chichkov et al., 1996; Loesel et al., 1998; Niemz, 2002).

Since femtosecond pulses are shorter than the thermal

diffusion time (picoseconds to nanoseconds), heat transport

is minimized, and the biological sample remains unaffected

by heat shock and subsequent damage (Loesel et al., 1998;

Niemz, 2002). This effectively renders the reversible perme-

abilization process non-thermal. Therefore, cell damage due

to thermal heating is inconsequential, demonstrating the

advantage of using ultrafast (femtosecond) laser pulses for

reversible permeabilization.

Future Prospects

The application of femtosecond lasers for non-invasive

reversible permeabilization of living cells provides an alter-

native method for achieving the biopreservation of engi-

neered and native cells and tissues. Since post-preservation

survival rates depend on the non-invasive nature of the

permeabilization tool, decreasing the degree of cellular

damage inflicted will inherently increase the preservation

efficiency rate. With the most widely used permeabilization

tools, like electroporation, transient pores irreversibly de-

nature and modify the functional groups of proteins (Tsong,

1991) and compromise cellular function. Therefore, femto-

second lasers provide a significant advancement over current

permeabilizaton techniques.

Furtherwork is being conducted on the post-cryopreserved

survival rate of mammalian cells permeabilized by ultrafast

laser pulses. We anticipate that our study will have important

applications in biopreservation, and expect that this new tool

will impact the preservation of more complicated biological

systems.

The authors would like to thank L.E. McGann and H.Y. Elmoazzen for

their technical expertise in transport equations and cell kinetics, and

H.Y. Elmoazzen, J. Lecak, K.L. Scott, and J.L. Anderson for their

review of this manuscript.

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