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Regen. Med. (2012) 7(3), 1–13
Research Article
10.2217/RME.12.7 © 2012 Future Medicine Ltd
Enhanced viability of corneal epithelial cells for
efficient transport/storage using a structurally
modified calcium alginate hydrogel
Transplantation of corneal epithelial cells is the
principal therapy for a leading cause of corneal
blindness, limbal stem cell deficiency (LSCD)
[1,2], which develops due to depletion of epithe-
lial stem cells at the limbal region of the cornea.
Currently, limbal stem cell transplantation is
undertaken in a few specialist hospitals across
the developed world. These cells are mainly
expanded ex vivo on amniotic membrane (AM)
in discrete laboratory facilities associated with
hospitals where the transplant is performed.
As techniques for limbal stem cell transplanta-
tion develop, hospital laboratories involved in
the production of cells for this therapy may be
replaced by larger distribution centers. Inherent
benefits of these centers include opportunities
to develop a broad base of expertise for cell
preservation and shipment and to replace and/
or compliment well-established but less efficient
and/or economical existing corneal tissue/cell
storage methods [3,4].
In the present study, we hypothesized that
biocompatible hydrogels may be applied to the
short-term transport/storage of corneal epithe-
lial cells that have been demonstrated to recon-
struct functional corneal epithelium in humans
presenting LSCD [1,2]. A clear advantage of this
method of cell storage in comparison with con-
ventional corneal tissue/cell storage methods [3,4]
is that it ensures the quick and easy retrieval of
suspended cells at their time of use.
Hydrogels have been applied extensively as cell
carriers or 3D cell culture systems [5–23]. Fibrin
hydrogels have been used for the delivery of lim-
bal epithelial cells (LECs) [5], and another bio-
material, electrospun poly(lactide-co-glycolide),
has been reported as a biodegradable cell carrier
system for these cells [6]. Collagen gels have also
been demonstrated as suitable substrates for the
expansion and delivery of LECs [7,8]. Alginate
hydrogels, one of the most well-characterized
cell immobilization substrates [9–15], have been
used to encapsulate a wide variety of cell types,
including retinal [9] and seminiferous tubule [10]
epithelial cells, probiotic bacteria [11], pancreatic
islet cells [12], parathyroid cells [13], neuroblas-
toma cells [14] and fibroblasts/keratocytes [15].
The immunoisolatry and semipermeable
structure of alginate gels has made this bioma-
terial an attractive substrate for the transplanta-
tion of cells as treatments for diseases including
hormone- (diabetes [12]) and protein-deficiency
conditions (hypoparathyroidism [13]). The struc-
tures of alginate gels have also been reported to
influence encapsulated cells and tissues [14,16–18].
Previous reports have demonstrated that an
increase in alginate gel porosity enhanced the
secretion of proteins and solutes from encapsu-
lated cells [16–18], and changing the alginate con-
centration has been demonstrated to influence
the movement of solutes through this gel [16].
The mannuronic and guluronic acid content of
alginate gels also determines permeability [19,20].
Alginate hydrogels have also been demon-
strated to direct cell phenotype, particularly
stem cell differentiation [24–30]. This biomaterial
Aims: Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated
batches were transported for 'on-demand' use. Materials & methods: In this study, corneal epithelial cell
viability in calcium alginate hydrogels was examined under cell culture in ambient and chilled conditions
for up to 7 days. Results: Cell viability improved as gel internal pore size increased, and was further
enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal
for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases
in pore size mediated by hydroxyethyl cellulose. Conclusion: Our novel methodology of controlling alginate
gel shape and pore size together provides a more practical and economical alternative to established
corneal tissue/cell storage methods.
Author Proof
KeywoRds: calcium alginate gel pore size n calcium alginate gel shape n calcium
alginate structure n corneal epithelial cell storage n corneal epithelial cell viability
n hydroxyethyl cellulose
Bernice Wright1,
Richard A Cave1,
Joseph P Cook1,
Vitaliy V Khutoryanskiy1,
Shengli Mi1,2, Bo Chen1,
Martin Leyland3
& Che J Connon*1
1Stem Cells & Nanomaterials
Laboratory, School of Pharmacy,
Hopkins Building, University of
Reading, Berkshire, RG6 6UB, UK
2Shaanxi Institute of Ophthalmology,
Xian, 710002, China
3Royal Berkshire Hospital, Reading,
Berkshire, RG1 5AN, UK
*Author for correspondence:
Tel.: +44 1183 787053
Fax: +44 8712 514665
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Regen. Med. (2012) 7(3)
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was previously reported to drive embryonic stem
cells toward definitive endoderm [27], as well as
to induce embryonic stem cell differentiation
into insulin-producing cells [28]. Mechanical
properties of alginate gels have been shown to
influence the differentiation of neural stem cells
[29]. Furthermore, the macrostructure of algi-
nate gels was shown to impact on the pheno-
typic characteristics of mesenchymal progenitor
cells [30].
The present study aims to demonstrate how
the structure of a calcium alginate gel may be
modified to allow corneal epithelial cells that are
immobilized within this scaffold to be preserved
in a viable state for short periods with limited or
no proliferation. Our approach using a hydrogel
system for the storage of corneal epithelial cells
in isolation has not been previously reported.
Published studies of the storage of these cells
have involved their ex vivo expansion on AM
before preservation in a variety of medium com-
positions under ambient conditions [31,32]. The
storage of corneoscleral buttons under chilled
(2–8°C) [3] and organ culture (28–37°C) [3,4]
conditions before their transplantation is estab-
lished – organ culture conditions are accepted
as the most suitable preservation methods for
this tissue [3,4].
We demonstrate differential viability profiles
for corneal epithelial cells stored in alginate gel
masses, which correlate with variations in the
sizes of gel internal pore spaces. Cell viability was
greatly improved upon modification of alginate
gels from an amorphous mass to a thin disc.
Furthermore, addition of hydroxyethyl cellu-
lose (HEC) as a live cell porogen significantly
augmented the viability of encapsulated cells by
increasing the size of internal pores within gels.
This study describes an alginate-based pres-
ervation method that may be applied to the
transport of corneal epithelial cells used in the
treatment of LSCD.
DMEM and Ham's F12 medium (DMEM/F12,
1:1), antibiotics (penicillin/streptomycin), fetal
bovine serum (FBS), amphotericin B, dimeth-
ylsulfoxide (DMSO), HCl, Whatman chroma-
tography paper, OCT (TissueTek) and Trypan
Blue were purchased from Thermo Scientific
HyClone (Fisher Scientific, UK). hEGF, insu-
lin, sodium alginate (viscosity: 15–20 cP, 1%
in H2O [lit.]), HEC (Mv 90 kDa, DS 1.5, MS
2.5), calcium chloride, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT
reagent), sodium citrate and sodium chloride
were obtained from Sigma-Aldrich (UK). The
human corneal epithelial (HCE) cell line was
from RIKEN BioResource Center (Japan).
Vectashield® containing DAPI was from Vector
Laboratories, Ltd (UK) and osmium tetrox-
ide was from Agar Scientific, Ltd (UK). The
enhanced chemiluminescence detection sys-
tem was obtained from Pierce (Thermo Fisher
Scientific, IL USA). The 14-3-3z antibody
was from Santa Cruz Biotechnology (Autogen
Bioclear UK, Ltd, UK), the CK3 antibody was
purchased from Millipore (UK) and the CK14
antibody was from Progen Scientific (UK).
Horseradish peroxidase-conjugated secondary
antibodies were from R and D Systems (UK)
and bovine serum albumin was obtained from
First Link (UK).
n Culture of a HCE cell line
A HCE cell line [33] was cultured in DMEM/
F12, 1:1, supplemented with 5% (v/v) FBS,
0.5% (v/v) DMSO, 10 ng/ml hEGF, 5 mg/ml
insulin, 100 IU/ml penicillin and 100 mg/ml
streptomycin, at 37°C under 5% CO2 and 95%
humidity. Cells were replenished with fresh
medium every 3 days and grown to 70–80%
n Isolation of epithelial cells from the
The established bovine cornea model [7,8,34] was
used for the isolation of LECs. Normal bovine
eyes were obtained from a local abattoir (Chity
Wholesale Abattoir, UK) within 2 h of death,
transported to the laboratory at 4°C and used
immediately. Corneoscleral rims were dissected
using standard eye bank techniques, as previ-
ously described [7,8,34].
n Encapsulation of epithelial cells in
calcium alginate gel masses & discs
HCE and primary LECs were suspended in
0.3% (w/v), 0.6% (w/v), 1.2% (w/v), 2.4% (w/v)
and 3.6% (w/v) sodium alginate solution with
or without 1.2% (w/v), 2.4% (w/v) and 3.6%
(w/v) HECs, before gelling into masses and discs
using 102 mM CaCl2. Gel masses and discs were
formed by pipetting 2 ml sodium alginate/cell
solutions into approximately 10 ml of 102 mM
CaCl2 and using chromatography paper moulds
(Whatman) immersed in 102 mM CaCl2,
respectively. Briefly, to form gel discs, a paper
ring with a 2-cm diameter opening was placed
over a 3-cm diameter paper disc. These were
saturated with 102 mM CaCl2 before alginate
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or alginate/HEC solution (420 µl) was pipetted
into the ring. A second 3-cm diameter paper disc
saturated with 102 mM CaCl2 was placed over
the alginate/paper assembly. Alginate or algi-
nate/HEC solutions were exposed to 102 mM
CaCl2 for 10 min to allow complete gelation.
Calcium alginate or alginate/HEC gel
masses and discs were suspended in supple-
mented DMEM/F12 medium under cell culture
(37°C, 5% CO2, 95% humidity) and ambient
(18–22°C, atmospheric CO2 and humidity
levels) and chilled (4°C, atmospheric CO2 and
humidity levels) conditions for 1, 3, 5, 7 and/
or 12 days. Primary LECs in alginate gels were
stored in DMEM/F12, 1:1, supplemented with
10% (v/v) FBS, 0.5% (v/v) DMSO, 10 ng/ml
hEGF, 5 mg/ml insulin, 100 U/ml penicillin and
100 mg/ml streptomycin. Initial experiments
performed under ambient and chilled conditions
using gels seeded with 5 × 105 cells/2 ml gel
demonstrated a rapid decline in cell viability,
which was prevented by reducing cell numbers
to 3 × 105 cells/2 ml gel (under cell culture stor-
age, gels were seeded with 5 × 105 cells/2 ml
gel). Periods of encapsulation were restricted to
7 days under ambient and chilled conditions, as
cells did not remain alive for longer periods of
time. Gel/cell matrices were replenished with
fresh medium every 2 days. Loss of cells from
calcium alginate gel masses due to the extraction
process was minimal, and nongelled alginate or
alginate/HEC did not significantly affect cell
n Cell viability analysis
Cells were extracted from calcium alginate gel
masses and discs using alginate-dissolving buf-
fer (0.15 M NaCl, 0.055 M sodium citrate).
Cells from individual conditions were cultured
in supplemented DMEM/F12 for approxi-
mately 3 days for HCE and 2 weeks for LECs
to monitor the ability of these cells to attach
and form colonies postextraction. Images of
cell colonies were obtained at 100× magnifi-
cation. The Trypan Blue exclusion assay was
performed by mixing a 10-µl cell suspension
with 10-µl Trypan Blue dye solution, before
counting live (unstained) and dead (stained
blue) cells using a hemocytometer. The MTT
assay was performed to assess cell metabolic
activity, following the manufacturer's proto-
cols. Briefly, 12 mM 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide was
added to a 100-µl suspension of cells, and this
was incubated for 2 h at 37°C. Cells were then
lysed using DMSO, incubated for a further
10 min at 37°C, mixed, and the absorbance
of the reaction product (formazan) was mea-
sured at 540 nm using an absorbance micro-
plate reader (model no.: 0112-0138; Molecular
Devices, Spain).
n Immunoblotting
Following the transfer of proteins to polyvinyli-
dene fluoride membranes, nonspecific binding
to membranes was blocked by incubation with
5% (w/v) bovine serum albumin dissolved in 1X
Tris-buffered saline–Tween® (TBS-T; 20 mM
Tris-base, 0.14 M NaCl, 0.1% Tween-20;
pH 7.6). Membranes were incubated with pri-
mary antibodies (anti-CK14 [1/8000 dilution]
and anti-CK3 [1/4000 dilution]) diluted in
2% (w/v) bovine serum albumin dissolved in
1X TBS-T at 4°C overnight. CK14 and CK3
expression were detected and blots were stripped
to remove primary antibodies before their total
levels were measured using anti-14-3-3z anti-
body (1/4000 dilution). Blots were washed
for 45 min in TBS-T before incubation with a
horseradish peroxidase-conjugated secondary
antibodies (antimouse [1:8000 dilution] and
antiguinea pig [1:10,000 dilution]) for 2 h at
room temperature with rotation. Proteins were
detected on x-ray film using an enhanced che-
miluminescence system.
n Scanning electron microscopy
analysis of calcium alginate structure
The internal surfaces of calcium alginate and
calcium alginate/HEC gels were examined by
scanning electron microscopy (SEM). Gels
were fixed in 1.25% (v/v) glutaraldehyde and
postfixed for 2 h in 1% (v/v) aqueous osmium
tetroxide; they were then washed in distilled
water and passed through a graded ethanol series
(50% [v/v], 70% [v/v], 90% [v/v] and 100%)
before dehydration through critical point dry-
ing. Dehydrated gels were fractured to expose
internal surfaces, mounted on aluminium stubs
and sputter-coated with gold before examination
using SEM (Quanta™ FEG 600; FEI, UK).
n Determination of alginate
mannuronic:guluronic acid ratios
Alginate solution was prepared in deion-
ized water at a concentration of 2.5% (w/v).
Alginate solutions (5-cm3 aliquots) were placed
in 10-cm3 glass microwave tubes (CEM) and
subjected to microwave irradiation using a
power input of 200 W, a nominal temperature
of 120°C and a hold time of 5 min. This method
was adapted from the microwave-assisted rapid
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hydrolysis method of Chhatbar et al. [35], by
using a research microwave reactor (CEM
Discover LabMate) instead of a domestic
microwave oven. The safety cut-off pressure of
200 psi was never reached. Following hydroly-
sis, the mannuronic:guluronic acid ratio (M:G)
was determined with reference to the method
of Chandía et al. [36]. Briefly, the hydrolyzed
solution was adjusted to pH 2.85 (monitored
with a Mettler Toledo SevenEasy pH meter
S20) using 0.1 M HCl (Fisher) solution and the
resulting precipitate, poly(guluronic acid) was
collected by centrifugation (Denley BS400)
and weighed. The supernatant was adjusted
to pH 1.0 using 0.1 M HCl solution and the
second precipitate, poly(mannuronic acid), was
collected by centrifugation and weighed. The
value obtained for the M:G ratio was 3.3 ± 0.3
(77% M:23% G), consistent with the manu-
facturer's assertion that this alginate has a high
M content.
n Analysis of the compressive
mechanical moduli of calcium
alginate/HEC hydrogels
Gel spheres were formed by dispensing 2 ml
alginate or alginate/HEC solutions dropwise
into 30 ml CaCl2 (100 mM). Gelled spheres
were mechanically tested at 1, 5, 10, 15, 20, 25,
30, 45 and 60 min to determine the time peri-
ods needed for complete gelation. Alginate and
alginate/HEC solutions gelled into stable gels
after 10–20 min exposure to 102 mM CaCl2.
Mechanical testing of gels was achieved by com-
pressing these structures using a TA.XT. plus
Texture Analyzer (Stable Micro Systems, UK)
with a 5-kg load cell and a P/1KS flat-ended
stainless steel probe (Stable Micro Systems) with
a 1-cm2 surface area. Measurement of the force
was taken, with a trigger force of 0.0005 N, and
the yield point of gel spheres (point at which the
gels split) was recorded. Force was recorded as
the mechanical limit of the gels, described as
comparative yield force, and ten measurements
of each gel sample were performed.
n Analysis of cell distribution in
HEC-modified calcium alginate
gel discs
HCE cells were encapsulated in calcium algi-
nate (1.2% (w/v) alginate) or HEC-modified
calcium alginate (1.2% [w/v] alginate/1.2%
[w/v] HEC, 1.2% [w/v] alginate/2.4% [w/v]
HEC) gel discs. Gels were embedded in OCT
solution, frozen and cryosectioned. Transverse
sections of gels were mounted on glass slides with
Vectashield containing DAPI fluorescent stain
to visualize cell nuclei. Sections were observed
by fluorescence microscopy (Carl Zeiss Meditec,
n Statistical analysis
One-way analysis of variance with Tukey post hoc
tests was performed using Prism for Windows™
– version 2.0 (GraphPad, Inc., CA, USA).
Results are presented as the mean of three indi-
vidual experiments with standard error of mean
and p ≤ 0.05 was considered significant.
n Calcium alginate supports corneal
epithelial cell viability in a differential
manner under various storage
To examine the suitability of a calcium alginate
hydrogel for preservation of corneal epithelial
cells, the effects of alginate concentration, stor-
age condition and period of encapsulation on
cell viability were investigated.
The viability of HCE cells (SV40-immortal-
ized cell line) within calcium alginate was
influenced by polymer concentration, period of
encapsulation and storage condition (Figure 1). Cell
viability was supported most effectively within
0.6% (w/v) alginate gel masses under all stor-
age conditions. Under cell culture storage after
a 5-day encapsulation period, encapsulated cells
(5 × 105) were more viable in a 0.6% (w/v) gel
mass (62 ± 11%) than in a 1.2% (w/v) gel mass
(25 ± 5%) (Figure 1A & 1D). Under chilled and
ambient storage, however, cell viability was sup-
ported less effectively. After a 3-day encapsulation
period, 57 ± 14% of initially encapsulated cells
(3 × 105) remained alive under chilled storage,
and only 33 ± 4% of cells recovered from ambient
storage were viable.
Postextraction culture of cells demonstrated
that cells from 0.3% (w/v) (Figure 1g, 1K & 1M)
and 0.6% (w/v) (Figure 1H, 1K & 1N) alginate gel
masses formed colonies, but cells from 1.2%
(w/v) gel masses (Figure 1i, 1L & 1O) did not attach
to culture dishes. Cells recovered from 0.3%
(w/v) gel masses (Figure 1g) formed smaller col-
onies than those from 0.6% (w/v) gel masses
(Figure 1H) maintained under cell culture stor-
age. This pattern of growth was particularly
apparent for cells from ambient (Figure 1J & 1K)
and chilled (Figure 1M & 1N) storage. Furthermore,
cells from cell culture storage formed larger col-
onies (Figure 1G & 1H) than those from ambient
(Figure 1J & 1K) or chilled (Figure 1M & 1N) storage.
Therefore, these data demonstrate that HCE
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Author Proof
Encapsulation period (days)
Live cells extracted
(% of total live cells encapsulated)
Encapsulation period (days)
Live cells extracted
(% of total live cells encapsulated)
Encapsulation period (days)
Live cells extracted
(% of total live cells encapsulated)
0.3% alginate
0.6% alginate
1.2% alginate
0.3% alginate
0.6% alginate
1.2% alginate
0.3% alginate
0.6% alginate
1.2% alginate
0.3% alginate
0.6% alginate
1.2% alginate
0.3% alginate
0.6% alginate
1.2% alginate
0.3% alginate
0.6% alginate
1.2% alginate
Encapsulation period (days)
Number of live cells extracted
Encapsulation period (days)
Number of live cells extracted
Encapsulation period (days)
Number of live cells extracted
100 µm100 µm 100 µm
100 µm100 µm100 µm
100 µm100 µm100 µm
Figure 1. The viability of human corneal epithelial cells stored in calcium alginate gel masses is dependent on polymer
concentration, period of encapsulation and storage condition. Data points represent the mean (n = 3 ± standard error of the
mean) percentage (A cell culture, B ambient and C chilled; 100% = initially encapsulated live cells) or number (d cell culture, e ambient
and F chilled) of live cells extracted. Images (n = 3; 100× magnification) show colonies of cells from cell culture (G 0.3% (w/v) alginate,
H 0.6% (w/v) alginate and I 1.2% (w/v) alginate), ambient (J 0.3% alginate, K 0.6% alginate and L 1.2% alginate) and chilled (M 0.3%
alginate, N 0.6% alginate and o 1.2% alginate) storage.
*p ≤ 0.05 indicate differences between alginate concentrations.
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cells can be stored most effectively under cell
culture conditions in a 0.6% (w/v) alginate gel
By contrast, the viability of physiological
primary LECs was supported most effectively
under chilled storage in 0.6% (w/v) alginate
gel masses (Figure 2A & 2B). The viability of these
cells (46 ± 5% cells alive) was, however, lower
than that of HCE cells (57 ± 14% cells alive)
maintained under chilled storage in 0.6% (w/v)
gel masses (see Figure 1C & 1F). Despite this reduc-
tion in viability, more than 50% of initially
encapsulated live cells were recovered after a
3-day encapsulation period within gels under
chilled storage (Figure 2A & 2B). A greater pro-
portion (50%) of live primary epithelial cells
was extracted from alginate gels under chilled
storage than from gels maintained under cell
culture storage (Figure 2A). Cells from cell cul-
ture storage, however, formed large sheets
postextraction (Figure 2C) whereas those from
chilled storage only formed small colonies
(Figure 2D), after extraction from gels. Under
ambient storage, LEC viability was very poor;
5 µm 5 µm5 µm
Figure 3. Internal pore network of calcium alginate gels. The internal surfaces of calcium
alginate gel masses (A 0.3% alginate, B 0.6% alginate and C 1.2% alginate) were examined using
scanning electron microscopy. Electron micrographs (19,000× magnification) represent three
individual experiments.
Encapsulation period (days)
Encapsulation period (days)
Number of live cells extracted
Live cells extracted
(% of total live cells encapsulated)
Cell culture
Cell culture
100 µm
100 µm
Figure 2. The viability of primary limbal epithelial cells stored in calcium alginate gel
masses is influenced by storage conditions. Data points represent the mean (n = 3 ± standard
error of the mean) percentage (A) (100% = initially encapsulated live cells) or number (B) of live cells
extracted. Images (n = 3; 100× magnification) show colonies of cells from cell culture (C) and chilled
(d) storage.
*p ≤ 0.05 indicate differences between storage conditions.
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cells did not remain alive for longer than 3 days
(Figure 2A & 2B).
Taken together, these data demonstrate that
0.6% (w/v) alginate gel masses store corneal epi-
thelial cells most effectively in either a chilled
or cell culture environment. These conditions
require further optimization, however, to limit
cell proliferation and enhance cell viability.
n The structure of calcium alginate
gels may influence the viability of
encapsulated cells
Variations in cell viability between gels formed
from different alginate concentrations (see
Figure 1) suggested that the structure of gels
may affect their ability to support viable cells.
Therefore, the internal structures of 0.3% (w/v),
0.6% (w/v) and 1.2% (w/v) alginate gel masses
were examined by SEM.
Gel masses comprised irregular internal pore
spaces that increased in size with decreasing
alginate concentration (Figure 3). Pore diameters
ranged from 0.2 to 3.0 µm within a 0.3% (w/v)
gel (Figure 3A), 0.1 to 1.0 µm within a 0.6% (w/v)
gel (Figure 3B) and 0.1 to 0.4 µm within a 1.2%
(w/v) gel (Figure 3C). Cells were more robustly
viable in 0.3% (w/v) and 0.6% (w/v) alginate gels
with large pores than 1.2% gels with relatively
Cell culture
Encapsulation period (days)
Number of live cells
extracted (×10,000)
Live cells extracted (% of
total live cells encapsulated)
(at 540 nm)
Encapsulation period (days)
Encapsulation period (days)
Cell culture
Cell culture
100 µm 100 µm100 µm
Figure 4. dimensions of calcium alginate gel masses and discs. Calcium alginate gel masses
(A & C) (0.6% alginate) are approximately 12.5 mm in length (A) and 6 mm in depth (C), and 1.2%
alginate gel discs (B & C) are approximately 19 mm in length (B) and 1.5 mm in depth (C). Images
(100× magnification) represent three individual experiments.
Figure 5. Limbal epithelial cell viability is enhanced within a thin disc of calcium alginate gel. Data points represent the mean
(n = 3 ± standard error of the mean) percentage (A) (100% = initially encapsulated live cells), number (B) or mitochondrial activity (C) of
live cells extracted. Images (n = 3; 100× magnification) show colonies of cells from cell culture (d), ambient (e) and chilled (F) storage.
*p ≤ 0.05 indicate differences between storage conditions.
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smaller pores (see Figure 1). Therefore, the inter-
nal structure of alginate gels may influence the
viability of encapsulated cells.
n The viability of primary epithelial
cells is enhanced in thin discs of
calcium alginate
Modifications to the shape of alginate gels have
previously been shown to enhance the viability of
immobilized neuroblastoma cells [14]. We there-
fore altered the macrostructure of gels from an
amorphous mass (~6 mm depth and 12.5 mm
width) to a thin disc (~1.5 mm depth and
19 mm width) (Figure 4). We hypothesized that
the reduced depth of a 1.2% (w/v) alginate gel disc
would compensate for low cell viability observed
in a 1.2% (w/v) gel mass by presenting a shorter
distance for mass transfer of nutrients to and waste
from immobilized cells, as previously reported [16].
Cell viability was enhanced in gel discs
(Figure 5) compared with gel masses (compare
Figures 2 & 5), and the number of live cells retrieved
from gels after storage did not exceed the number
of initially encapsulated live cells. In gel discs,
the highest proportions of viable cells were
recovered from ambient storage (Figure 5A & 5B),
which was previously demonstrated as unable to
support viable cells in 0.6% (w/v) gel masses for
longer than 3 days (see Figure 2). After a 3-day
encapsulation period, under ambient storage,
a significantly greater (p ≤ 0.05) proportion of
initially encapsulated cells remained alive in gel
discs (43 ± 7%) (Figure 5A & 5B) than in gel masses
(18 ± 2%) (Figure 2A). After a 7-day encapsula-
tion period, 52 ± 5% of initially encapsulated
cells (3 × 105) remained alive in gel discs under
ambient storage (Figure 5A). After 1-, 3- and 5-day
encapsulation periods, however, cell viability
between gel masses and discs were similar under
chilled storage (compare Figures 2 & 5).
As observed within gel masses (see Figure 2),
the lowest levels of viable cells were extracted
from gels maintained under cell culture storage
(Figure 5A & 5B). In gel discs, live cells extracted
from cell culture storage were, however, able
to form cell sheets (Figure 5D). This pattern of
postextraction growth was similar to that of live
cells from ambient storage (Figure 5g). Although
cell viability was robust in gel discs under chilled
storage, during postextraction culture, only sin-
gle cells from chilled storage adhered to culture
dishes (Figure 5F). The mitochondrial activities of
cells from chilled and ambient storage were simi-
lar (Figure 5C). Therefore, the inability of chilled
storage live cells to form colonies or sheets was
not due to reduced levels of metabolically active
The phenotype of LECs encapsulated in algi-
nate gels was also influenced by storage condi-
tions. Both differentiated and undifferentiated
LECs were demonstrated to be present in 1.2%
(w/v) alginate gel discs under cell culture, ambi-
ent and chilled storage after 5 days (Figure 6).
Under chilled conditions, a marker for undif-
ferentiated LECs, CK14 (Figure 6A), was expressed
to similar levels as CK3 (Figure 6B), the specific
marker for differentiated LECs. CK3 expression
was greater than CK14 expression in LECs stored
under cell culture and ambient conditions.
Collectively, these data demonstrate that
modification of the macrostructure of a calcium
alginate gel from an amorphous mass to a thin
disc enhances corneal epithelial cell viability,
limits cell proliferation and overcomes the reduc-
tion in cell viability observed with increases in
alginate concentration.
n Modification of internal pore sizes
of alginate gel discs with HEC
significantly enhances corneal
epithelial cell viability
We demonstrated that increases in the size of
internal pores in alginate gels correlate with
increases in cell viability (compare Figures 1 & 3).
Therefore, to further enhance cell viability,
methods were employed to increase pore sizes
in alginate gel discs. HEC was demonstrated to
enlarge the size of internal pores within these
gels (Figure 7) in the presence of live cells.
Cell viability was markedly increased in algi-
nate gel discs formed in the presence of HEC
compared with gel discs without this polysaccha-
ride (Figure 8 & suppLeMeNtAry Figure 1) www.future-
medicine.com/10.2217/rme.12.7). A 1.2% (w/v)
gel disc supported 60 ± 11% of encapsulated cells
in a viable, state while modification of these gels
ChAmC CCCh Am
Figure 6. differentiated and undifferentiated limbal epithelial cells are
maintained in alginate gels under cell culture, ambient and chilled
conditions. CK14 (A) and CK3 (B) were detected in freshly isolated limbal
epithelial cells (C condition) and limbal epithelial cells stored for 5 days in alginate
gel discs under CC, Am and Ch conditions by immunoblotting. 14-3-3z was used as
a loading control. Blots represent three individual experiments from three different
corneoscleral rims.
Am: Ambient; C: Control; CC: Cell culture; Ch: Chilled; MW: Molecular weight.
Author Proof
Page 9
Enhanced viability of corneal epithelial cells using a calcium alginate hydrogel Research Article
future science group
with 1.2% (w/v) or 2.4% (w/v) HEC maintained
the viability of 100% (p ≤ 0.05) of encapsulated
cells (Figure 7). Improved cell viability correlated
with a decrease in gel stiffness (compressive
modulus) (Figure 8), as well as increased pore
size (Figure 7). Cells in 1.2% (w/v) alginate gel
discs modified with HEC were more dispersed
than those in 1.2% (w/v) gel discs without this
nonionic polysaccharide (Figure 9), suggesting
that cell distribution may also be affected by gel
structure. Gels comprising 2.4% (w/v) or 3.6%
(w/v) alginate supported low levels (20–40%)
of live cells (Figure 8) compared with 1.2% (w/v)
alginate gels. Cell viability in 2.4% (w/v) and
3.6% (w/v) alginate gels modified with HEC
was not improved, despite increases in pore
sizes (Figure 7). This suggests that the increased
stiffness of these gels may overcome increases in
viability due to enlarged pore size.
These data indicate that the viability of cells
encapsulated within calcium alginate gels is
influenced by both pore size and mechanical
properties. The extent to which these structural
features affect cell viability may be manipulated
by modifying proportions of alginate and poro-
gen (HEC) in gels.
The transport of live cell cultures presents a
significant distribution problem for laboratory
supply companies and emergent therapeutics
companies specializing in regenerative medicine.
Current distribution solutions mainly involve
cryopreservation and/or cold chain transport,
which are impractical, economically costly and
technically challenging, as they either require spe-
cialized logistics or can only be performed in lim-
ited time-delivery windows [101]. Alternatives to
these conventional methods for cell distribution
may improve the clinical management of thera-
peutic cells, as well as contribute favorably to the
early promise of cell-based regenerative medicine.
A hydrogel that is structurally stable at a range of
temperatures, biocompatible for cell encapsula-
tion and easily modified to quickly release encap-
sulated cells (calcium alginate) would be a suitable
alternative that may be developed for significant,
wide-ranging use in future cell-based therapies.
The present study therefore investigates cal-
cium alginate as a storage module for corneal
epithelial cells. The structural dependence of the
biocompatibility of this hydrogel is examined.
Modifications to the macrostructure, internal
microarchitecture and mechanical properties of
alginate hydrogels have been previously reported
to influence the behavior of immobilized cells
[14,16–18]. We hypothesized that altering gel shape,
pore size and mechanical properties together may
enhance the biocompatibility of alginate hydro-
gels for use as substrates for corneal epithelial
cell storage.
Effective transport of solutes in and out of
cell immobilization scaffolds (i.e., alginate
hydrogels) is an essential property to ensure
their biocompatibility [37,38]. In the present
study, modifications to alginate gel shape and
internal pore size possibly increased the viability
of immobilized cells by causing more efficient
mass transfer of nutrients and oxygen to, and
metabolic waste from, cells by viscous flow [39]
through enlarged pores. An increase in alginate
5 µm
5 µm
5 µm
5 µm
5 µm
5 µm
Figure 7. The internal pore network of hydroxyethyl cellulose-modified
calcium alginate gel discs. Calcium alginate (A 1.2% alginate, C 2.4% alginate
and e 3.6% alginate) and hydroxyethyl cellulose (HEC)-modified calcium alginate
(B 1.2% alginate/2.4% HEC, d 2.4% alginate/2.4% HEC and F 3.6%
alginate/2.4% HEC) gel discs were dehydrated and internal surfaces were examined
using scanning electron microscopy. Electron micrographs (20,000× magnification)
represent three individual experiments.
Author Proof
Page 10
Research Article
Wright, Cave, Cook et al.
Regen. Med. (2012) 7(3)
future science group
gel porosity was reported to enhance ammo-
nia elimination rates of hepatocarcinoma cells
[17] and albumin secretion by hepatocytes [18].
Therefore, HEC-mediated increases in alginate-
encapsulated cell viability, which were observed
in the present study, may be due to the displace-
ment of alginate polymer by HEC, leading to the
enlargement of forming pores.
The permeability of alginate gels has also
been shown to be dependent on alterations in gel
shape [14] and gel stability [16]. Increasing alginate
concentrations from 1.5 to 3% was previously
shown to hinder the movement of various sol-
utes through this gel, as 3% alginate gel internal
pore diameters were shorter (147 Å) than 1.5%
alginate gel internal pore diameters (170 Å) [16].
Alginate gel permeability is also determined by
their M and G content [19,20]: gels with a high
M:G ratio present a small intrinsic pore size [40].
In the present study, the alginate used comprised
a high M percentage (77%) and a low G percent-
age (23%). The M:G ratio may be manipulated
to increase the permeability of gels as necessary
to improve the viability of encapsulated cells.
High M alginates are, however, more biocom-
patible and clinically useful than high G algi-
nates [20,40]. Therefore, the method described in
this study that incorporates HEC into a high
M alginate gel to modify the porosity of this
biomaterial is advantageous, as the gel retains
the high elasticity and low viscosity conferred
by the high M content.
Cell storage in alginate gels has not been previ-
ously investigated alongside the structure of this
biomaterial. Reports investigating cell storage in
alginate gels have, however, examined the viabil-
ity of a number of different cell types in alginate
gels under both ambient and chilled storage. In
the present study, ambient storage was the most
favorable condition for LECs in alginate gels.
Similarly, LECs expanded on AM in organ cul-
ture medium have been demonstrated to retain
high levels of viability under ambient storage [31].
Spermatozoa stored under ambient conditions
in alginate gels were reported as more function-
ally robust compared with those not stored in
alginate gels [41]. Neuroblastoma cells stored
in alginate gels at 4°C were reported to retain
HEC concentration (% [w/v])
Yield force (N)
Alginate concentration
(% [w/v])
Figure 8. Corneal epithelial cell viability in hydroxyethyl cellulose-modified alginate gel
discs correlations with the mechanical properties of gels. Data points for cell viability represent
the mean (n = 3 ± standard errorof the mean) percentage (100% = initially encapsulated live cells) of
live cells extracted. Data points for compressive moduli represent ten individual measurements.
HEC: Hydroxyethyl cellulose.
Author Proof
Page 11
Enhanced viability of corneal epithelial cells using a calcium alginate hydrogel Research Article
future science group
executive summary
Need for effective methodologies for therapeutic cell transport
Currently, cells are mainly distributed using cryopreservation and/or cold chain transport, which are impractical, economically costly and
technically challenging.
Future cell distribution centers could be designed to develop cell preservation and shipment methodologies, which could replace less
efficient and economical corneal tissue/cell storage methods.
Response to need for cell transport methods
An alginate-based preservation method that can be applied to the transport of corneal epithelial cells was investigated.
Alginate hydrogels with modifications to the macrostructure, internal microarchitecture and mechanical properties were examined as
cell transport vehicles.
Corneal epithelial cell viability in structurally modified calcium alginate hydrogels was examined under cell culture, ambient and chilled
Suitability of alginate hydrogels as cell transport vehicles
Alginate hydrogels are structurally stable at a range of temperatures and are easily modified.
These gels are biocompatible for cell encapsulation, and encapsulated cells can be quickly released.
Alginate hydrogels are biocompatible, as the porous structure of these scaffolds allows effective transport of solutes to and from
immobilized cells.
200 µm
200 µm
200 µm
Figure 9. The distribution of cells in alginate and hydroxyethyl cellulose-
modified calcium alginate gel discs. Cells within calcium alginate (A 1.2%
alginate) or hydroxyethyl cellulose (HEC)-modified calcium alginate (B 1.2%
alginate/1.2% HEC and C 1.2% alginate/2.4% HEC) gel discs containing human
corneal epithelial cells were visualized using DAPI fluorescent nuclei stain (n = 3).
Arrows indicate cell dispersion.
normal morphological differentiation and adhe-
sion abilities [42], and oocytes stored at 4°C were
also demonstrated to be functional when restored
to physiological environments [43]. By contrast,
chondrocytes stored at 4°C in alginate gels were
only modestly viable [44].
In the present study, the recovery of live corneal
epithelial cells from chilled (4°C) storage was also
compromised. These cells did not form colonies
when extracted from alginate gels. This may be
because cells were undergoing anoikis, which is
programmed cell death that prevents cell attach-
ment [45]. Corneal epithelial cells have been previ-
ously reported to undergo apoptosis under chilled
conditions [46]. It may be possible to achieve com-
plete recovery of live cells stored under chilled
conditions by incorporating CO2 into alginate
gels, as the viability of cells stored in alginate gels
under chilled conditions has been reported to
improve through normalization of increased pH
with CO2 [44]. The storage of corneoscleral but-
tons under chilled conditions has been successful
[3]; therefore, encapsulation in alginate gels may
improve the preservation of epithelial cells within
these tissues under chilled conditions.
The structure of alginate hydrogels has also
been reported to influence the stem phenotype
of encapsulated progenitor cells [29,30]. In the
present study, alginate gels maintained epithe-
lial cells in both undifferentiated and differenti-
ated states. Previous studies have demonstrated
that alginate hydrogels control the differentia-
tion of stem cells [29] and that the macrostruc-
ture of alginate gels can determine the extent
of cell differentiation [30]. Therefore, the 1.2%
(w/v) alginate gel disc used in the present study
potentially comprises structural characteristics
conducive to maintaining the stem phenotype
of limbal epithelial progenitor cells.
This study provides the first line of evidence
demonstrating how both the macro- and micro-
structure of a calcium alginate hydrogels can be
modified to support the short-term preservation
of corneal epithelial cells.
Conclusion & future perspective
We have evolved alginate encapsulation tech-
nology by describing structural modification of
this biomaterial for potential use as a transport/
storage device applicable to corneal cell-based
Author Proof
Page 12
Research Article
Wright, Cave, Cook et al.
Regen. Med. (2012) 7(3)
future science group
Papers of special note have been highlighted as:
n of interest
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Author Proof
The authors would like to thank C Stain (University of
Reading, Centre for Advanced Microscopy, UK) for providing
technical assistance.
Financial & competing interests disclosure
The work was funded by the Medical Research Council
(MRC; Grant Ref.: G/0900877). The authors have no
other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or finan
cial conflict with the subject matter or materials discussed
in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
ethical conduct of research
The authors state that they have obtained appropriate insti
tutional review board approval or have followed the princi
ples outlined in the Declaration of Helsinki for all human
or animal experimental investigations. In addition, for
investi gations involving human subjects, informed consent
has been obtained from the participants involved.
Page 13
Enhanced viability of corneal epithelial cells using a calcium alginate hydrogel Research ArticleResearch Article
future science group
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text:
Chris Diamantopoulos Celebrity Watchlist
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Pronunciation: tifosi
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A Duchy of Lothringen-Alsace would make for a nice Buffer-state, solve that whole France/German 'its ours' debate and make Xavier mad XD
A Duchy of Lothringen-Alsace would make for a nice Buffer-state, solve that whole France/German 'its ours' debate and make Xavier mad XD
Syndicalism defeated. Yay!!!
general Otto Metzler in a Federation of Equals
Maximilien Bellamy in "Edge of Europe"
Leave Deutschland be, attack Japan, and release:
Basque Country (Basque Carlists)
Portugal (with Galicia to punish Spain)
Aquitaine (Occitan or Southern France)
Wales + Cornwall
Rest of UK
United Italy
Well, if Remble is secretly controlling Japan he would thrash you.
In Canonized's Timelines: What if Spain Failed to Control the World?, awarded 20 points for pointing out the centrifuge used at one point; identified a cell phone; detected the inspiration for a bounty hunter; inspired a Cube scene and given 30 points for a Star Wars reference.
Solved (with help from Davout) Canonized's Puzzle Plates Challenge
Fan of the week August 24, 2008.
Given 20,000 cookies by Maj. von Mauser.
Has two ships named after him in DvD-IT's Das war ein Befehl! and a carrier in TRP's The Channel Pact
Awarded the Pizza of Iron, First Class, in Kurt Steiner's Timelines: What if Stauffenberg had failed to save the world?
Won a Cookie of GLORY in Alexus' Impo- Difficult Victory: A Japan Mod34 VE/A AAR
Well done! I see you have also imported some of the US election events from the vanilla game.
I like that; the most irritating thing about the US in the normal Kaiserreich is that there are no
elections after 1936.
Please no Balkanization nonsense.
Keep France and Spain united. They'll make powerful allies and nice puppets.
Awarded Fan of the Week 10. April 2010
Awarded the Medal of Services or Knowledge towards the betterment of Canada
But what can be done with the small part of France which Germany has conquered? If you check this map of late medieval France (Link) you'll notice that the French land held by Germany in this story corresponds indeed to the 15th century territories of Artois and Picardie, and the Duchy of Champagne.
If the Germans feel secure enough to piss off the French, they could just turn it into a protectorate and give it a medieval sounding name. The local people will hate it, and probably wipe their asses with the flag whenever the Germans are not looking. But it would make for a nice new principality for one of the many side lines of the Hohenzollern family. (The Al-Sauds of the Kaiserreich world.)
Enslave the evile Syndie French population under the iron fist of the exiles in Algiers. That will teach them a lesson.
general Otto Metzler in a Federation of Equals
Maximilien Bellamy in "Edge of Europe"
Awarded Fan of the Week 10. April 2010
Awarded the Medal of Services or Knowledge towards the betterment of Canada
Uh people he's playing a democratic US not some dictatorial fascist state run by a nutjob. Return Great Britain and France to their respectful rulers, then release Portugal, Spain, and Italy as republics.
I am glad to see so many comments after the end of the war against syndicalism. It is good to know that everyone liked it.
Anyway, I wanted to share with you all an idea I had about what the Germans could do with their occupation zone. Although making little kingdoms and duchies would be interesting, it is not really realistic for the 20th century. Therefore, here is my idea: the Franco-Dutch Federation/Union.
This new country could become a very powerful ally to the Germans, which with the end of the war have taken control of not only a portion of northwestern france but also the netherlands and flanders-wallonia. This new state could be ruled either from Amsterdam or Brussels, and would be led by the long exiled Dutch royal family, with both Dutch and French government ministers. This new state would have a pretty good industrial base, and could provide a very secure buffer between Germany and the Entente.
In Defense of Freedom, A US Kaiserreich AAR Complete. Awarded Weekly AAR showcase, March 15th, 2010
Rebirth of the Japanese Empire, A Japan MDS 1.6 AAR Complete, Awarded Weekly AAR showcase, February 5th, 2012
The Return of The King: A Canadian KaiserReich DH AAR Complete, Awarded Darkest HeAARt Award, April 2014
The Dual Monarchy at War A 1914 Austria-Hungary DH AAR Complete, Awarded Weekly AAR showcase, March 9th, 2014
Freedom's Darkest Hour, a Customized USA Modern Day Scenario AAR In Progress
Awarded WritAAR of the week: February 14th, 2010
"All the great things are simple, and many can be expressed in a single word: freedom, justice, honor, duty, mercy, hope."
Winston Churchill
"I like your Christ. I do not like your Christians. Your Christians are so unlike your Christ."
Mohandas Gandhi
Far better it is to dare mighty things, to win glorious triumphs, even though checkered by failure, than to rank with those poor spirits who neither enjoy much nor suffer much, because they live in that grey twilight that knows neither victory nor defeat.
Theodore Roosevelt
I like it.
Your life is your life dont let it be clubbed into dank submission.
Be on the watch there are ways out.
You cant beat death but you can beat death in life, sometimes.
And the more often you learn to do it the more light there will be.
Your life is your life.
Know it while you have it.
The gods wait to delight in you.
You should clean up the borders a little bit.
"Politics. As exciting as war. Definitely as dangerous... Though in war, you can only get killed once. In politics it can happen over and over."
Give Troyes to France - use the rivers as a boundary IIRC the Germans didn't actually take that unless they grabbed it at the end. If they did have it as 'Germany shows it's magnaniminity' and hand it over to France. Give (Chaumont? Whatever, the one above Dijon) to Germany,


text:
Coated Tongue After Touching Precum Worried I Have HIV
Here's a taste of what TheBody.com has to offer on this topic:
Get the Facts
What Are the Symptoms of HIV and AIDS?...an HIV-positive woman unprotected oral sex (if you're providing the mouth; this is a much lower risk than the others) That's it. For any other type of activity -- kissing someone with HIV, sharing food or drinks with them, touching something an HIV-positive person touched -- there's basically no chance that whatever you did resulted in you getting HIV. If you're curious about whether specific sexual acts are riskier than others, check out...
Ask the Experts
Intestinal gas/coated tongue HIV or something else?You are doing a great job. We thank you. please answer my question I beg you. I had sex from October till February. On November and February I had constipation and rashes on my groin area, which are gone now. Now I have coated tongue from February...
Response from Ryan M. Kull, C.S.W
Columbia University's Health & Related Services...medical doctor would have to do that in person. This is way out of my league because I do not think this question relates to HIV or STDs.Best of luck.RMK Read more »
Egads! Coated Tongue or what?Hey Doc!First off, thanks for all you tireless work on this site as well as HIVDENT. A quick question for you... about a month after a risky exposure, I noticed ONLY the back of my tongue was coated with a thin layer of tartar-like paste, which I...
Response from David A. Reznik, D.D.S.
Grady Health System...hairy tongue. It is very common for coated and tongue to become stained by the foods you eat, so in this regard I would not worry.You know this part of the response if coming........... If you are concerned about your HIV serostatus, the only way... Read more »
MESSAGE FOR THE WORRIED WELLDear Robert,Here's something for the worried well, which was my category.I was absolutely convinced I had HIV. I had overall bodily itching, looked it up on the web and sure enough itching is an HIV symptom. Then my doctor informed me that I had...
Response from Robert J. Frascino, M.D.
The Robert James Frascino AIDS Foundation...internship, residency, post-doctoral fellowship, and sub-specialty training?Thank you for your donation. I assure you it will touch many lives. On behalf of those folks, as well as personally, please accept my sincere appreciation.Stay well. I... Read more »
1 year later and still coated tongueDr. Reznik14 months have passed since my seroconversion illness and i still present with coated tongue, some days worse than others. I have been to a dentist of ENT doc who said just to brush tongue. This problem is quite irritating. Since my VL...
Response from David A. Reznik, D.D.S.
Grady Health System...Dear Jack:Coated tongue is not related to HIV infection per say. In other words, to my knowledge there is nothing regarding the pathogenesis of HIV infection Read more »
High Anxiety: AIDS Anxiety in the Worried-Wells and Worried-Sicks, Part Two...progression and drug resistance; changes in body shape, appearance and libido; fear of infecting others -- some worry even their touch has become toxic; fears surrounding stigmatization and discrimination; health insurance; career plans; job... Read more »
High Anxiety: AIDS Anxiety in the Worried-Wells and Worried-Sicks, Part One...Revenge of a disgruntled baggage handler Toothbrush spunk follow-up question-Will donate Nov 8, 2009 Want to get in touch with Dr. Bob? You can reach him through his "Ask the Experts" forum , by sending a message to the Robert James... Read more »
Philippines Worried About Rapid Rise in HIV/AIDS Cases... Responding to a worrying spike in new HIV/AIDS diagnoses, the Philippines health department today vowed to increase its outreach to those most at risk. Read more »
First Person: An Interview With Shelley Singer...you." Then he stuck me in a room. Two of his hygienists come in, and they are in gowns, gloves and masks. Now, they're not touching me at all, but they are in gowns, gloves and masks. They went around that little exam room, and they emptied it... Read more »
Connect With Others
worried of getting stabbed with hiv in my foot
Posted by worried1234, 3 Replies...but me and my friend didnt really bother about them at all,and then they came back after a while from the other direction,they touched me and my friend's hips and walked off,one of them turned around sticking his or her tongue out at us i am... Read more »
Positive for Hep C and now worried about HIV
Posted by Worrywart28, 2 Replies...exception of 2 things. My hiv results hadn't come in but she said this is normal because they have to mail that test. Still, that worries me. She also told me i tested "reactive" to Hepatitis C. My range was very low though. Anything over 1.00 is... Read more »
worried I've got HIV after oral without
Posted by anonymouslad, 6 Replies...be nothing nothing to do with a possible std infection. But this along with regularly feeling mildly sick is getting me very worried I'm one of the tiny tiny percent of people who've been unlucky enough to get HIV through unprotected. Do these... Read more »
Long-term HIV and Aids survivors' health worrying
Posted by ScotCharles...From KaiserNet January 8, 2008 Some experts and doctors recently have voiced concerns that people who were diagnosed with HIV and Aids in the early years of the epidemic are experiencing "premature[e]" or "disproportionate numbers" of ailments... Read more »
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Main News Page
Friday, August 12, 2011
More items for
Quality Control
Supersonic rain experiments and other leading-edge protective coatings testing will continue at the University of Dayton Research Institute, thanks to a new $24.5 million contract from the U.S. Air Force.
The five-year contract allows the university's researchers to test and evaluate protective paints and coatings for aerospace and other applications.
The responsibility includes continued operation and maintenance of the Air Force Research Laboratory's Coatings Technology Integration Office, Special Test and Research facilities, and water- and sand-erosion labs at Wright-Patterson Air Force Base. The university team has operated the facility for 10 years.
Under the new contract, the Research Institute also will operate AFRL's new Supersonic Rain Erosion (SURE) facility, which will shoot raindrops at speeds up to Mach 2.5 to test the durability of coatings, sealants and other aerospace materials. The new contract replaces and expands on a recently expired five-year, $10.1 million contract.
Through Rain, Sand and Extremes
The UDRI Paint Testing and Coatings Research Group evaluates coatings and solves paint problems for government and industry. The group, which includes 17 coating professionals, is accredited to ISO 17025 and SAE AS 5505 for the application, testing, and performance characterization of organic coatings.
The group's largest customer is the Air Force CTIO at Wright-Patterson. The office is the Air Force's central resource for aircraft coating systems and their applications.
University researchers test and evaluate the performance of coatings and materials used primarily for military and commercial aerospace applications under a variety of conditions, including extreme temperatures and humidity; they also assist in the development of advanced coatings.
In the erosion labs, researchers use simulated rainfall, sand and other materials and processes to test the durability of coatings exposed to water erosion and particle erosion.
Developing the Right Paint Stuff
"Paints, sealants and other coatings contain special properties that protect surfaces from damaging environments," said Bill Culhane, coatings group leader at the institute.
"By scientifically evaluating coatings, we can verify they are doing their jobs and recommend the best coatings for specific applications and environments. It's much easier and [more] efficient to protect a surface with the right paint than to replace the surface," Culhane said.
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Aware of the rout it will face in Seemandhra, the Congress has pinned its hopes on a pre-election tie-up with the TRS and one after the polls with Jaganmohan Reddy
No one is in doubt that political considerations, more specifically the anxiety to grab the most number of Lok Sabha seats in Telangana, have made the Congress fast track the partitioning of Andhra Pradesh.
Not much imagination is needed either to predict that the Congress will face an unprecedented rout in coastal Andhra and Rayalaseema where it does not even stand a remote prospect of winning any one of the 25 Lok Sabha seats. It is thus forced to put all its electoral eggs in the Telangana basket. The anti-Congress wave now is an extreme swing from the popularity of "Indiramma" whose pro-weaker sections image guaranteed the party a minimum 30 per cent votes in every election, win or lose, since the 1970s.
What defies logic or compulsions of realpolitik is that the Congress has firmly shut the door on itself in the race for the 25 Lok Sabha seats in Seemandhra while running at breakneck speed to breast the tape in the 17 constituencies in Telangana. There is no guarantee that it will win a majority of seats in Telangana without a pre-poll alliance with the Telangana Rashtra Samithi (TRS)'s K. Chandrasekhar Rao who is playing hardball.
All eyes on this team
Yet, throwing caution to the winds, the Centre is racing ahead with the division of Andhra Pradesh. The State's future now hangs by a slender thread in the form of the seven-member Group of Ministers. None of them are from Andhra Pradesh and belong to places as far as Kashmir (Ghulam Nabi Azad) and Kerala (A.K. Antony). They will sit and divide the Telugus, their water resources, their State capital, their revenue and, what many fear, their hearts and minds. They are mandated to fulfil in six weeks a task that took the Srikrishna Committee one full year, only for its report to be thrown into the waste-paper basket.
YSR Congress leader Jaganmohan Reddy has accused Congress president Sonia Gandhi of dividing Andhra Pradesh with the objective of installing her son, Rahul Gandhi as Prime Minister. While this may sound a bit far-fetched, what may be true is that the Congress party's prime minister-in-waiting may find it difficult to realise his dream without Jagan's support after the Lok Sabha election — he is considered the front runner in Seemandhra. Did he not sweep the Assembly byelections last year by massive margins?
In this context, much significance is being read into the statements of Digvijay Singh, AICC general secretary in-charge of Andhra Pradesh, a friend of the late YSR and considered his bridge with Ms Sonia Gandhi. A few examples are: "Congress and YSR Congress share the same DNA" and "Jaganmohan Reddy is like my son."
It may all sound like a cosy relationship — a pre-poll alliance with the TRS and post-electoral adjustment with the YSR Congress. But what the Congress does not realise is that it takes two to tango, politically. The Bharatiya Janata Party (BJP), already a partner of the TRS, is being wooed by the Telugu Desam's N. Chandrababu Naidu who, during the past one month, shared a platform with Gujarat Chief Minister Narendra Modi, and called on BJP president Rajnath Singh.
Sense of betrayal
Quite surprisingly, Jaganmohan Reddy, in a slight change of tack, described Mr. Modi as an able administrator though with a caveat that he would like the BJP's prime ministerial candidate-in-waiting to pursue secular policies. The Rashtriya Swayamsevak Sangh (RSS), though a votary of smaller States, is unhappy with the BJP, for turning its back on Seemandhra where its cadre and supporters have deserted the party en masse.
Many people outside Andhra Pradesh may not know Vizianagaram except old-timers who remember Vizzy the famous cricket commentator on radio, who hailed from there. It is a town with a low profile, famous earlier for royal patronage of the arts, literature and higher education. Its institutions turned out famous film playback singers Ghantasala and P. Susheela while the town produced great social reformer Gurazada Appa Rao.
When Vizianagaram exploded into violence last weekend leading to police firing and the imposition of curfew, it was symbolic of the seething anger in Seemandhra over the Cabinet's perceived insensitivity to the people's concerns. The suffering caused by stoppage of RTC buses since August 12, the ruination of the micro-level economy, bandhs and power blackouts were inconsequential.
All that mattered was their anger that the Centre had reneged on its promise to redress fears about their children's education, their jobs, availability of water for irrigation and the safety of their kin living in Hyderabad, a city that would ultimately be handed over to Telangana. Worse still, Union Ministers from Seemandhra and leaders, particularly Vizianagaram-based APCC chief Botcha Satyanarayana, were seen as conniving with the Centre while the A.K. Antony Committee meant to redress their grievances had vanished into thin air.
The sense of betrayal in Seemandhra is so deep that Congress leaders are gravitating either towards the YSR Congress or the Telugu Desam while some are taking temporary retirement from politics to focus on expanding their business empires. The BJP cannot be oblivious to the extent to which the political class as a whole has alienated itself from the people during the two-and-a-half month long agitation that was totally people-driven. In fact, it isn't now since the party has been criticising the way the Centre has ignored the concerns of Seemandhra while pressing ahead with bifurcation.
It would be interesting to see if the main Opposition party, which has backed the United Progressive Alliance on several occasions in the monsoon session, would go along with the Congress on Telangana too when the Constitution Amendment Bill comes up in Parliament for passage by two-thirds majority.
Keywords: Andhra Pradesh Bifurcation, Telangana statehood, Congress party, TRS, Jagan Mohan Reddy, K. Chandrasekhar Rao


text:
Stephen Vincent Benet
Alternate Georges
On the birthday of the Father of Our Country it is proper to take a moment and reflect that in all likelihood the United States of America would not exist today but for the leadership shown by George Washington during the Revolution. The poets Rosemary and Stephen Vincent Benet explored long ago some of the many different paths the life of Washington might have taken which would have altered our history so profoundly. We call Washington the Father of Our Country not to honor him, but as a simple statement of fact. Continue reading
February 6, 1862: Surrender of Fort Henry
Fate has a way of picking unlikely material,
Greasy-haired second lieutenants of French artillery,
And bald-headed, dubious, Roman rake-politicians.
Her stiff hands were busy now with an odd piece of wood,
Sometime Westpointer, by accident more than choice,
Sometime brevet-captain in the old Fourth Infantry,
Mentioned in Mexican orders for gallant service
And, six years later, forced to resign from the Army
Without enough money to pay for a stateroom home.
Turned farmer on Hardscrabble Farm, turned bill-collector,
Turned clerk in the country-store that his brothers ran,
The eldest-born of the lot, but the family-failure,
Unloading frozen hides from a farmer's sleigh
With stoop-shouldered strength, whittling beside the stove,
And now and then turning to whiskey to take the sting
From winter and certain memories.
It didn't take much. A glass or two would thicken the dogged tongue
And flush the fair skin beneath the ragged brown beard.
Poor and shabby–old "Cap" Grant of Galena,
Who should have amounted to something but hadn't so far
Though he worked hard and was honest.
A middle-aged clerk,
A stumpy, mute man in a faded army overcoat,
Who wrote the War Department after Fort Sumter,
Offering them such service as he could give
And saying he thought that he was fit to command
As much as a regiment, but getting no answer.
So many letters come to a War Department,
One can hardly bother the clerks to answer them all–
Then a Volunteer colonel, drilling recruits with a stick,
A red bandanna instead of an officer's sash;
A brigadier-general, one of thirty-seven,
Snubbed by Halleck and slighted by fussy Frémont;
And then the frozen February gale
Over Fort Henry and Fort Donelson,
The gunboats on the cold river–the brief siege–
"Unconditional surrender"–and the newspapers.
Stephen Vincent Benet
The taking of Fort Henry by Ulysses S. Grant on February 6, 1862, was important for a number of reasons:
1. It opened the Tennessee River to Union gunboats and transports down through northern Alabama, effectively allowing the Union to outflank Confederate
defenses in Memphis and throughout eastern Tennessee. Continue reading
Sam Grant, the Beatles and the Internet
I feel that we are on the eve of a new era, when there is to be great harmony between the Federal and Confederate. I cannot stay to be a living witness to the correctness of this prophecy; but I feel it within me that it is to be so. The universally kind feeling expressed for me at a time when it was supposed that each day would prove my last, seemed to me the beginning of the answer to "Let us have peace."
Ulysses S. Grant, written just before his death
Something for the weekend. Quotations from Ulysses S. Grant to the Beatles song In My Life. A follow up to my post on Robert E. Lee, the Beatles and the Internet. Another demonstration of what a wild and wacky place the internet truly is!
Few men in American history have gone from complete obscurity to being a central figure in the life of the nation faster than Ulysses Simpson Grant. Known as Sam Grant by his West Point friends, his first two initials making Sam an inevitable nickname, Grant had an unerring ability to fail at everything he put his hand to, except for war, his marriage and his last gallant race against the Grim Reaper, as he was dying of cancer, to finish his memoirs and provide financially for his wife and children. Most great figures in our history have known success more than failure. Not so Sam Grant. He would encounter humiliating defeats throughout his life, from beginning to end.
At the beginning of the Civil War, he was a clerk, barely able to support his family. Seemingly a dull plodder, but possessed of iron determination and an uncanny ability to never let the trees obscure the forest; happily married and a firm believer in God, but subject to bouts of depression when he would grasp for the bottle; the shabby little man who, incredibly, ended up winning the greatest war in American history.
His men didn't hold him in awe as Lee's men did Lee; Grant was far too common and prosaic a figure for that. However, they did respect him, as this section of Stephen Vincent Benet's epic poem on the Civil War, John Brown's Body, indicates: Continue reading
United States v. Satan
A scene from the classic movie, The Devil and Daniel Webster, based upon the short story by Stephen Vincent Benet, in which Daniel Webster bests Satan in a jury trial to save the soul of New Hampshireman Jabez Stone. Prior to the trial, Daniel Webster attempts to get Jabez Stone out of the contract on the ground that the Devil is a foreign prince. Satan denies this:
Foreign!" said the stranger. "And who calls me a foreigner"? "Well, I never yet heard of the dev?? of your claiming American citizenship," said Dan'l Webster with surprise. "And who with better right?" said the stranger, with one of his terrible smiles. "When the first wrong was done to the first Indian, I was there. When the first slaver put out for the Congo, I stood on her deck. Am I not in your books and stories and beliefs, from the first settlements on? Am I not spoken of, still, in every church in New England? 'Tis true the North claims me for a Southerner and the South for a Northerner, but I am neither. I am merely an honest American, like yourself and of the best descent for, to tell the truth Mr . Webster, though I don't like to boast of it, my name is older in this country than yours." "Aha!" said Dan'l Webster, with the veins standing out in his forehead. "Then I stand on the Constitution! I demand a trial for my client!"
This story is actually cited in a federal legal opinion which may be read here, and which is riotously funny in a very dry sense. I submit it to establish that some judges do have a sense of humor, at least of a sort.
Returning to the short story, I have always treasured this passage: the closing argument of Daniel Webster to the Jury of the Damned, which I think contains wisdom about patriotism and the human condition: Continue reading
Marse Robert
Some of our readers south of the Mason-Dixon line no doubt have perhaps felt left out in my many posts regarding Abraham Lincoln. I am fully aware that great Americans fought on both sides of the Civil War, and one of the greatest of Americans, of his time or any time, was Robert E. Lee.


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Cabin By The Sea Is A 3/2.5 Townhouse In The Gated River Cove Community. The Townhome Features Sweeping Views Of The River From Every Room. The Home Is Two Stories Over A Covered Parking/storage. The First Floor Is The Kitchen, Living Space, Half Bath And Master Bedroom (tiled Floors) And The Second Floor Is Two Guest Bedrooms And Full Bathroom (carpet). The River Cove Community Is Less Than One Mile From The Public Boat Ramp And Four Miles From Catching Beautiful Sunsets At Fort Island Park Or, Launching Your Boat, Canoe Or Kayak To Explore The Gulf Or The Salt Marsh Trails. Most Restaurants And Grocery Stores Are Less Than A 10 Minute Drive. By Boat, From The Nearby Boat Ramp, The Gulf Is 10 Minutes In One Direction And Kings Bay Is 10 Minutes In The Other Direction. Cabin By The Sea Is The Perfect Place To Stage Your Visit To Crystal River. Canoes, Kayaks And Paddle Boards Can Easily Be Stored In The Covered Carport And Launched From The Shore Behind The Townhome Giving You Quick Access To Salt Marshes, The Gulf And The Springs Of Kings Bay. Homosassa River And Springs Is A 15 Minute Drive Away And Within 30 Minutes You Can Access The Withlacoochee River And Crystal Clear Water Of Rainbow River. Surrounding Crystal River Are Numerous State Parks Ideal For Hiking, Bird Watching And Canoeing Or Kayaking. The Flats Of The Gulf Are A Fisherman's Paradise And Snorkeling The Springs With The Manatees Of Crystal River Is Must For Families Of All Ages.
We purchased Cabin By The Sea to get away from the hustle of our week day lives.
Dan McLean purchased this townhome in 2013
Crystal River is an amazing town. It has the necessities of a large city but the feel and hospitality of a small town. And, you can't beat the location. EVERYTHING is only a short drive or boat.
Springs, river, Gulf... the environment diversity is unmatched. There is something for everybody.
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I think just about any additional scenes to SUPERMAN:THE MOVIE just make it better and even more enjoyable.
Go ahead and buy this version of the DVD with confidence, it's a great transfer with good sound and the extra footage is a nice bonus.
The acting is good, the action is plentiful, and the special effects are great for the time period it was made.
If anyone out there has not seen this great film yet, I'd be surprised. So, we are talking about seeing it again, or seeing it on Blu-Ray for the 1st time. Read morePublished 3 days ago by Beantown Jim
This still manages to be greatest Superman movie of all time and one of the superhero movies of all time. So much amazing qualities. Reeve is a godsend as Superman. Read morePublished 6 days ago by James Jones
Before the doomed planet Krypton explodes, Jor-El and wife Lara send their infant son, Kal-El, to Earth to save his life. Read morePublished 6 days ago by A.P. Fuchs, author of The Axiom-man Saga
I don't show my son comic cartoons because I don't appreciate all the violence and special effects that so unrealistic to our world, but I wanted him to know who is this Super Man... Read morePublished 17 days ago by Evgenia Loshinina
shipped on time..what can i say great blast from the past in blu ray.. must own for any collectorPublished 28 days ago by Mark
Disk 1. The Original 1978 movie----(digitally restored), Disk 1. a 2008 re-edited version, Disk 3. lots of documentaries, and
Disk 4. an old TV version. Read more
|From this Discussion
|Why does reversing the rotation of the Earth make time go backwards?
Speeding up would only make time for you go slower than people on earth but it would still be going forward. Even Einstein never figured out how to go backward in time. It was just for effect and fun. It never bothered me but it does kinda amaze me than in a film where a man flies and has x-ray... Read More
Jul 23, 2010 by Cornhusker | See all 12 posts
|Why does Marlon Brando wears the "S" on his chest at the beginning of...
The "S" is the family symbol for the House of El! Remenber to us it is an "S" but it is in fact a Kryptonian glyph. Think ancient egyptians or sumerians.
Jan 2, 2013 by J. RAMIREZ | See all 5 posts
|Problems with Superman DVD's
I love Superman I. It was great in the theater. Then, it was sold to TV many years ago, at which time about 45 minutes was added to the film that knocked my socks off again leaving me to wonder why these scenes were not part of the film to begin with. Now the original release was about 128... Read More
Mar 21, 2012 by Ray Sutton | See all 4 posts
|Deleted scenes
your lucky,that was the original release version when they first aired it over 30 years ago on abc, i wish i had that version, although i do have superman 2 on vhs when abc first aired it, like in the mid 80s, it has 14 minutes of extra footage not on the theatrical release, the superman dvd has... Read More
Mar 2, 2012 by Donny | See all 3 posts
|Superman 2 was better.
I like Superman 2, but I love the first one. It is my favorite superhero film and even one of my favorite films in any genre. I think it is a miracle of a film. It works because of the incredible cast, great dialogue and a dual performance by Christopher Reeve that is just flawless. He really did... Read More
Mar 15, 2012 by Cornhusker | See all 6 posts
|Which version is the BD: 1978 release cut (Donner's fav), or the 2000...
|Be the first to reply


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Comfort Inn
333 Great Northern Rd.
Sault Ste. Marie, ON P6B 4Z8
Phone: (705) 759-8000
Fax: (705) 759-8538
Sault Ste. Marie Walk of Fame
The Walk of Fame is to honor prominent personalities who have made an immense difference to local region or the entire globe. In the past, the recipients were Walter Newman, John Barker, Phil Esposito and so forth. For a comprehensive list, please refer the website.
269-289 Queen Street East
Sault Ste. Marie, ON P6A
Earn valuable points toward free nights.


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REVIEW: Dierks Bentley Makes A Pit Stop in NYC for IHeartRadio
The whirling dervish that is the Dierks Bentley promo tour for his new album Home made a quick pit stop in NYC yesterday for some lucky IHeartRadio contest winners. Dierks played a full show on Wednesday at the Bowery Ballroom but Monday night's 10 song, 45 minute set was a nice appetizer. The tiny P.C. Richards Theater on the ground floor of the Clear Channel building was packed with lots of Dierks' faithful fans – and they were not disappointed. Calling NYC the pulse of this country and bantering about talking country music with a man from Kentucky over breakfast in the Big Apple, Dierks Bentley knows that NYC knows country music! The showcase highlighted some of the new tracks including 5-1-5-0, a Van Halen inspired track, the title track Home, and When You Gonna Come Around featuring Karen Fairchild from Little Big Town which is a sweet, romantic, country ballad. Mrs. Fairchild was not in attendance last night but the song held up just fine as a solo number too. The fan favorites were peppered in as well. The audience was participating for Feel the Fire and What Was I Thinking. But when Dierks came down off the stage and into the crowd for Sideways the fan love soared! My personal favorite of the night was Up On The Ridge, the title track from Dierks' previous album. It may have been because Brooklyn's own Punch Brothers played on the album track or it may have been because it is an awesome song. But any which way you cut it last night made me proud to be from NYCountry.
This review was written by a guest writer, Erin Kamenitz, who attended the show on behalf of Keepin' It Country Blog.
Photo Credit: Jennifer Pottheiser for iHeartRadio
Jessica Crans
Latest posts by Jessica Crans (see all)
- Ram Trucks and Miranda Lambert Kick Off Brand Partnership - November 14, 2014
- First Ever Country Music Festival, FarmBorough, Set For NYC In June 2015 - November 10, 2014
- Chris Young Partners With Kidde to Increase Fire Safety - November 10, 2014


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STEP BY STEP FAMILY SUPPORT CENTER Reviews
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Step by Step provides many services outside of the traditional maternity/infant case management.
They have a Christmas program which feeds and gifts 100's of families. Once you are a client you are always welcome at any of their programs.
A free garage sale yearly. They also have a housing program, and life skills classes
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We would look 3 recent sold comps in the neighborhood, 3 active listings, and take in to consideration relevant updates to the home, attempting paint an accurate picture for you what the fair market value of the home is. Of course, many purchase and sale contracts offer the opportunity for an appraisal contingency. This aids to make sure that a buyer doesn't pay more than the house is worth.
I hope this helps.
As always, you can message me directly and I would also be glad to discuss.
Thank you.


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Corvallis 104.3 FM • Hood River 91.9 FM • Portland 90.7 FM
President Obama announced last night that he plans to make immigration reform a top priority during his first year in office.
KBOO's Jenka Soderberg spoke with local immigrant rights activist Francisco Lopez about what this pledge could mean for Oregon's immigrant community:
The Obama Department of Justice has put forth a claim of
"Sovereign Immunity" in a motion to dismiss in the domestic
spying case of Jewell v. NSA. The lawsuit brought by the
Electronic Frontier Foundation is suing the National
Security Agency for spying on tens of thousands of citizens
without a warrant, during the Bush Administration. The
spying utilized the telecom company AT&T's fiber optic
network to access the communications of unsuspecting
Americans. The claim of Sovereign Immunity is unprecedented
Dark Horse Comics president Mike Richardson returns to Words & Pictures to preview the 2009 Stumptown Comics Festival, running April 18-19 at the Lloyd Doubletree Hotel. The festival is preceded by a benefit dinner on April 17 for the Comic Book Legal Defense Fund.
Mayor Sam Adams has declared April to be Portland Comics Month in honor of the strong base of independent talent making their home in Oregon. Mike is joined by comics artist and PNCA instructor Neal Skorpen, who is leading a collaborative workshop at Stumptown on The Instant Graphic Novel.
A number of local anti-war veteran groups have called on the Portland City Council to declare the city a 'sanctuary' for war resisters.
The group is currently collecting signatures for a petition to submit to the City Council, which has not yet taken any action on the 'Sanctuary City' proposal.
Host Glen Andresen discusses space-saving techniques for growing vegetables in the city.
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KANSAS CITY, Mo. (AP) -- Seven were not enough for Diane and David Petersohn, so they decided to adopt.
Because one of their seven children has Down syndrome, a type of disorder caused by a genetic malfunction, they decided they wanted another one with that condition.
The Petersohns found that they had plenty of company. The couple from Liberty, Mo., placed their names on national lists of people seeking to adopt children with Down syndrome, and waited.
After waiting nearly three years, they turned to a private agency that facilitates international adoptions. Today, they're raising money and completing paperwork to adopt a 6-month-old boy from Ecuador who has the syndrome.
Most who seek to adopt children with Down syndrome have had a family member, friend or acquaintance with that chromosomal disorder, or work with them in medical or school professions.
"People think they are just great kids; people feel like they are very lovable," said Rachel Crews, a social worker with the Special Additions adoption agency in Stillwell, Kan.
Changing attitudes are helping unite these children with families, advocates say.
"Society as a whole is much more accepting," said David Tolleson, executive director of the National Down Syndrome Congress in Atlanta. "You are much more likely today to see people with disabilities in the media, places of worship, schools. Whereas in a prior generation, mothers were told ... [to] put the child in an institution and forget about them."
That's what happened 34 years ago to a little girl named Martha, whose single mother gave her up for adoption. Martha was diagnosed with Down syndrome and placed in a group home in Cincinnati.
But when Martha turned 4, Robin Steele and her husband met her and fell in love immediately. With one son already, they adopted Martha and have gone on to adopt nine other children -- three of them with Down syndrome.
Martha's adoption also spurred the Steeles to start the Adoption Awareness Program in conjunction with the Down Syndrome Association of Greater Cincinnati, which connects people who want a child with Down syndrome with biological mothers or adoption agencies.