Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF ISOLATING MESENCHYMAL STROMAL CELLS AND
APPLICATIONS FOR TISSUE ENGINEERING
Field of Invention
The invention relates to a method of isolating mesenchymal stromal cells,
particularly
for use in research following subsequent expansion and differentiation thereof
Background
Mesenchymal stromal cells are stem cells which are able to develop into a
variety of
cell types, although as they are pluripotent (as opposed to totipotent), not
an embryo
that could develop into an organism.
As a result, mesenchymal stromal cells are the subject of much research, and a
good
source of the same is highly desirable.
However, when isolating mesenchymal stromal cells from the source material
there
remain inconsistencies in the current best practise isolation techniques,
which result in
consumable wastage. This causes increased production cost. Microbial
contamination
during isolation is also a common occurrence as is the production of
substandard
mesenchymal stromal cells.
An aim of the invention is therefore to provide an isolation method which
overcomes
these difficulties.
Summary of Invention
In an aspect of the invention there is provided a method of isolating
mesenchymal
stromal cells comprising the steps of:
washing a tissue sample at least 3 times;
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incubating the tissue sample in a solution comprising at least one antibiotic
and
at least one antimycotic at a temperature of around 28-38 C and atmosphere
comprising
about 4-6% CO2;
washing the tissue sample with phosphate buffered solution to substantially
remove antibiotic and antimycotic therefrom;
cutting the tissue sample into small pieces;
incubating the tissue sample in collagenase dissolved in DMEM-KO at a
temperature of around 28-38 C and atmosphere comprising about 4-6% CO2 to
produce
a solution of tissue fragments;
centrifuging the solution of tissue fragments to form a pellet, which is
resuspended and transferred into MSC culture media for subsequent incubation;
characterised in that MSC culture media is added to the tissue sample during
the
collagenase incubation to quench the action of the collagenase such that the
tissue
sample is only partially digested.
Advantageously this produces cells with better viability (83-89% prior to
cryopreservation) compared to prior art methods involving complete digestion,
whereby
the resulting cells are obtained by straining the completely digested tissue -
complete
digestions result in poor viability and cells which are biologically stressed.
An
.. additional advantage is that significantly less consumables are required.
In one embodiment the MSC culture media is added when the tissue sample is
transparent but still substantially solid and not yet liquefied by the
collagenase.
In one embodiment the initial washing of the tissue sample is with sterile
distilled water.
In one embodiment the at least one antibiotic comprises penicillin and
streptomycin in
PBS.
In one embodiment Trypsin-EDTA is added to the solution of tissue fragments
and
incubated at 28-38 C, 4-6% CO2 for around 5 minutes to detach cells from the
matrix.
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In one embodiment the tissue fragments in MSC culture media are incubated in a
low
oxygen hypoxia or CO2 incubator for about 24 hours to promote growth of cells.
Typically the MSC culture media is repeatedly replaced until the cells reach
about 70-
80% confluency.
In one embodiment the cells are expanded through the following steps:
washing with PBS;
adding a Trypsin-EDTA solution and incubating at 28-38 C, 4-6% CO2 to detach
cells;
adding MSC culture media to the cells;
centrifuging the cells then resuspending the pellet in MSC culture media;
seeding the cells into MSC culture media
incubating in a low oxygen hypoxia or CO2 incubator until the cells reach 70-
80% confluency, replacing the MSC culture media every 2-4 days or so.
Typically the expansion steps can be repeated several times to progress the
cells from
passage 0 to passage 3, wherein a final product is formed on resuspending the
pellet.
In one embodiment the final product undergoes cryopreservation.
In one embodiment the tissue sample is derived from umbilical cord, foreskin
or cornea.
Typically the arteries, veins and blood components are removed from the
umbilical cord
while minimising the loss of the Wharton's jelly therefrom. Advantageously
this
decreases contamination by erythrocytes, as well as any other cells, cell
fragments or
derivatives thereof from umbilical vein (eg HUVEC). The prior art method does
not
involve complete removal of vein and arteries and also results in accidental
removal of
the matrix substance which produces decreased/inferior cell yield.
In one embodiment the umbilical cord is processed in about 3-5 separate
segments.
Advantageously this minimizes contamination, provides replicate samples, and
reduces
the consumables required due to the higher surface area:volume ratio. The
prior art
methods involve the whole umbilical cord being processed as a single sample
whereby
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erythrocytes, fibrinogen, fibrin, cell debris, cell contents etc., from a
region of the cord
can act as contaminants during tissue incubation which accelerates microbial
contamination.
.. With regard to umbilical cord, the tissue sample is typically incubated in
about 1-5%
(w/v), preferably around 3% (w/v) collagenase type IV for approximately 10-12
hours.
With regard to foreskin, the tissue sample is typically incubated in about 1-
5% (w/v),
preferably around about 1-5% (w/v), preferably around 3% (w/v) collagenase
type IV
for approximately 4-10 hours.
With regard to cornea, the tissue sample is typically incubated in about 1-5%
(w/v),
preferably around 3% (w/v) collagenase type IV for approximately 2-4 hours.
.. In one embodiment the tissue sample is dental pulp. Typically in this
embodiment the
initial washing of the tissue sample is with Dulbecco's phosphate buffered
saline
without calcium or magnesium.
In one embodiment the dental pulp is extirpated from a freshly extracted
deciduous
tooth. Typically the tooth is sectioned horizontally at the cementoenamel
junction using
a sterilized diamond disc while being irrigated with saline or distilled
water.
In this embodiment the tissue sample is typically incubated in about 1-5%
(w/v),
preferably around 3% (w/v) collagenase type I for about 30 minutes.
In one embodiment the incubation of tissue samples is conducted at a
temperature of
around 37 C and atmosphere comprising about 5% CO2
In a further aspect of the invention, there is provided a tooth holder
comprising:
a circular rotating chuck fixed to a base;
the chuck including three jaws and a collar, whereby rotation of the collar
extends or retracts the jaws thereby adjusting the aperture size between the
jaws to
allow a tooth to be held thereby;
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irrigation means above the chuck for dripping liquid onto the tooth;
characterized in that the chuck includes a central channel through which
liquid
may be pumped.
5 In one embodiment the jaws are provided with grooves or steps to allow
for precise
measurement and placement of the tooth.
In a further aspect of the invention there is a provided a system for
immunophenotyping
mesenchymal stromal cells comprising the steps of:
extracting total RNA from a sample of cells;
synthesising cDNA from the RNA;
determining the expression of genes using semi-quantitative RT-PCT analysis
by amplifying the cDNA using two or more primer pairs;
characterised in that the genes and respective primer pairs are selected from
SEQ
ID Nos: 1 and 2; 3 and 4; 5 and 6; 7 and 8; and/or 9 and 10.
Expression of these genes, which are dopaminergic neuronal markers, indicates
that the
cells are mesenchymal stromal cells capable of creating a dopaminergic cell
line that
can be used a research grade product e.g. for Parkinson's disease research.
Typically the sample of cells is a final product.
In a further aspect of the invention there is provided a tissue or product
made by
differentiating mesenchymal stromal cells produced by the method as herein
described.
In a further aspect of the invention there is a provided a dopaminergic cell,
neuron or
derivative thereof made by differentiating mesenchymal stromal cells produced
by the
aforementioned method.
In a yet further aspect of the invention there is a provided corneal tissue
made by
differentiating corneal limbal cells derived from mesenchymal stromal cells
derived
from a cornea tissue sample according to the aforementioned method.
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Typically cornea tissue can be grown in different layers to form a cornea.
In a yet further aspect of the invention there is a provided skin tissue made
by
differentiating mesenchymal stromal cells derived from a foreskin sample
according to
the aforementioned method.
This skin tissue may be used for foreskin restorative surgery or human skin
grafts, e.g.
for use in treating burn victims.
Brief Description of Drawings
It will be convenient to further describe the present invention with respect
to the
accompanying drawings that illustrate possible arrangements of the invention.
Other
arrangements of the invention are possible, and consequently the particularity
of the
accompanying drawings is not to be understood as superseding the generality of
the
preceding description of the invention.
Figure 1 illustrates different types of mesenchymal stromal cells in the
respective
finished products.
Figure 2 illustrates the ability of different types of mesenchymal stromal
cells to form
aggregates/neurospheres in NIM media.
Figure 3 illustrates the results of semi-quantitative RT¨PCR analysis of the
expression
of certain genes for immunophenotyping of mesenchymal stromal cells
Figure 4 illustrates immunocytochemistry of different types of mesenchymal
stromal
cells
Figure 5 illustrates a tooth holder according to an embodiment of the
invention
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Detailed Description
The techniques for isolating stromal cells from mammalian umbilical cord
(Wharton's
Jelly), dental pulp, foreskin and cornea are described below.
ISOLATION OF WHARTON'S JELLY STROMAL CELLS
Transporting the umbilical cord upon delivery
Umbilical cord should be clamped ideally within 30 to 60 seconds following
birth of the
infant placed. The clamp is placed at or below the level of the placenta to
allow optimal
blood transfer from the placenta to the infant. Sterile surgical blades or any
other sterile
cutting instruments or implement are used to cut the umbilical cord cut into
three to five
segments and placed into 50m1 polypropylene conical centrifuge tubes pre-added
with
25m1 of transport buffer stored at 4 C, 5 C, 6 C, 7 C, 8 C, 9 C, 10 C. This
entire
process should be done in a labour room with temperature ranging from 20 C to
25 C
and the umbilical cord must be placed into the transport buffer within 24
hours upon
birth to maintain tissue viability. The umbilical cord is then transported to
the laboratory
for mesenchymal stromal cell isolation process. Components of transport buffer
are as
described in Table 1 below.
Table 1
No. Transport Buffer Components Final concentration
1. Sodium chloride (NaCI) 133 to 140mmol/L
2. Potassium chloride (KCI) 2.4 to 3mm01/L
3. Sodium phosphate (Na2HPO4) 9.5 to 10.5mmo1/L
4. Potassium phosphate (KH2PO4) 1.5 to 2.0mmol/L
5. Antibiotic-Antimycotic* 5 to 12%
6. Penicillin-Streptomycin* 5 to 12%
7. Sterilized distilled water 0.8 to 1.2L
8. Hydrochloric acid (36%) 0.2 to 1000m1 until pH of transport buffer
reach 7.2 to 7.5 at 20 C to 25 C
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Umbilical cord cleaning process
The umbilical cord is processed in a Class II Biological Safety Cabinet (BSC)
with
following environment conditions; room temperature (25 C 5), relative humidity
(65% 10). The following process is carried out entirely in a BSC sterilized
with 70%
ethanol and 30 minutes UV radiation. The 50m1 polypropylene conical centrifuge
tubes
containing segmented umbilical cords are wiped with 70% ethanol. The following
apparatus and materials in Tables 2-3 below are used for the cleaning process.
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Table 2
No. Apparatus/material Sterilization method Quantity/
Volume
1. Surgical scissors (sharp/sharp) Autoclave (121 C/20-30 minutes) 5
2. Surgical scalpels Autoclave (121 C/20-30 minutes) 5
3. Surgical
blades Gamma radiated 15
4. 50m1 polypropylene conical centrifuge Gamma radiated 32
tubes
5. Micropipettor
(10-100pL) 70% ethanol & UV radiation 1
6. Micropipettor
(100-1000pL) 70% ethanol & UV radiation 1
7. Micropipette tips (2-200pL) Autoclave (121 C/20-30 minutes) 96
8. Micropipette tips (50-1000pL) Autoclave (121 C/20-30 minutes) 96
9. Surgical tray Autoclave (121 C/20-30 minutes) 5
10. Sterile
serological pipettes (5m1) Gamma radiated 15
11. Sterile
serological pipettes (10m1) Gamma radiated 15
12. Sterile
serological pipettes (50m1) Gamma radiated 15
13. Sterilized distilled water (SDW) Autoclave (121 C/20-30 minutes) 2L
14. Pipettor
70% ethanol & UV radiation 1
15. Phosphate buffered saline (PBS) Autoclave (121 C/20-30 minutes) 2L
16. Antibiotic-
Antimycotic (100X)* Sterile filtration (0.2pm) 10%v/v
17. Penicillin-
Streptomycin (100X)* Sterile filtration (0.2pm) 10%v/v
18. Collagenase
type IV* Sterile filtration (0.2pm) 3%w/v
20. Ethanol Not
required 70%v/v
21. Cell culture
plate (60mm) Gamma radiated 15
22. DMEM-KO*
Sterile filtration (0.2pm) 97%v/v
23. Trypsin-EDTA
(0.25%)* Sterile filtration (0.2pm) 2%v/v
24. MSC culture
media (see Table 3) Sterile filtration (0.2pm) 1L
25. Tissue forceps Autoclave (121 C/20-30 minutes) 7
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Table 3
No. MSC culture media components Final Volume (ml) Concentration
(%)
1. DMEM-KO 880
80 to 90
2. Anti biotic-
Antimycotic 5 0.2 to 1
3. Penicillin-
Streptomycin 5 0.2 to 1
4. Fetal Bovine Serum
(FBS) 100 5 to 12
5. GI utamax 10
0.5 to 1.5
Upon transportation of the umbilical cord to the laboratory, the cord is
segmented into 5
parts and the segmented cord is transferred into five individual 50m1
polypropylene
5 conical centrifuge tubes pre-added with 30m1 SDW. Segmented cords are washed
by
inverting the tubes repeatedly to remove blood traces to prevent blood
contamination.
This step is repeated at least three times to ensure the cords are thoroughly
washed
using fresh SDW and 50m1 polypropylene conical centrifuge tubes.
10 The segmented cords are transferred to the surgical tray to remove blood
clots and
traces of any components contained within blood including but not limited to
neutrophils, lymphocytes, plasma, and erythrocytes. This is to prevent blood,
blood
related products, and any other products from causing contamination. Umbilical
cord
contains two umbilical arteries and one umbilical vein, both embedded within a
specific
mucous proteoglycan-rich matrix which is known as Wharton's Jelly, which is
then
covered by amniotic epithelium. The umbilical arteries and vein are carefully
removed
by means of dissection without losing the matrix substance in which most of
the
Wharton's Jelly Stem Cells (WJSCs) are found. WJSCs can be isolated from three
regions of the umbilical cord, namely, the perivascular zone, the inter-
vascular zone and
the sub-amnion. The cord is then immersed in 70% ethanol for 25 to 30 second
to
sterilize the surface and immediately immersed into SDW for another 25 to 30
seconds
to remove ethanol residues and create an immediate hypotonic environment on
the
cord's surface to eliminate microbes.
The cords are then transferred into five individual 50m1 polypropylene conical
centrifuge tubes pre-added with 25m1 10% v/v Antibiotic-Antimycotic and 10%
v/v
Penicillin-Streptomycin in PBS. The tubes are incubated at around 28-38 C,
about 4-6%
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CO2 for around 2-16 hours, preferably 37 C, 5% CO2 for 2 hours/overnight. To
minimize contamination, the umbilical cord is processed as five individual
segments
until the end of the isolation process. The incubator is programmed at around
28-38 C,
about 4-6% CO2, preferably at 37 C, 5% CO to provide an optimal environment
for the
cord tissues/cells to maintain viability.
Wharton's jelly stromal cell isolation process
Prior to incubation, each segmented cord is washed with PBS twice for 30 to 60
seconds, to remove traces of Antibiotic-Antimycotic and Penicillin-
Streptomycin. The
cords are cut into small pieces (2-5mm) and transferred to a fresh 50m1
polypropylene
conical centrifuge tubes containing 20m1 of 3% w/v collagenase type IV
dissolved in
DMEM-KO. Cords are allowed to be partially digested at around 28-38 C, about 4-
6%
CO2 for around 2-16 hours, preferably 37 C, 5% CO2 for 10 to 12 hours. Then
400 1 of
0.25% Trypsin-EDTA is added into the partially digested cord and incubated at
37 C,
5% CO2 for 5 minutes to detach cells from the matrix. To neutralize the
enzymatic
action, 20m1 of MSC culture media is added into each tubes and centrifuged at
about
1500-2500rpm, 700-1000g force/RCF, for around 8-12minutes, at about 25-28 C,
preferably about 1800rpm for 10 minutes at 25 C. It is crucial to neutralize
the
enzymatic action as cell viability will reduce if Trypsin is allowed to stay
in contact
with the cells for too long. Prior to centrifugation, 50% of the supernatant
is carefully
removed by pipetting to eliminate excess MSC culture media and Trypsin.
Undigested semi-transparent umbilical cord fragments from the tubes are picked
using
sterile tissue forceps and transferred into 60mm cell culture plate. The
process is
repeated till all umbilical cord fragments are transferred and left to air dry
for 15minutes
in the BSC. This step is to ensure the tissues are attached to the culture
plate surface.
Upon adherence of the cord tissue fragments onto the culture plate, 5-7m1 of
MSC
culture media is added gently without disrupting the attached tissues. The MSC
culture
media provides sufficient nutrients and an optimal environment for the
survival of the
tissues. Culture plates are then transferred to a CO2 incubator or low oxygen
hypoxia
incubator and incubated for around 16-36 hours, preferably about 24 hours.
After 24
hours incubation, 2/3 of the media together with some matrix substance are
pipetted out
gently without disrupting adhered tissues and replaced with fresh MSC culture
media.
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The culture plates are transferred back to the CO2 incubator and an additional
5m1 of
fresh MSC culture media is added in the next 24 hours for continuous supply of
nutrients for cell growth and also to remove dead or apoptotic cells from the
culture.
The culture is observed using an inverted microscope every day and 2/3 of
media is
replaced every 2 to 4 days until the cells reach 80% confluency until
subcultured to
passage 1 stock. Around 70-80%, preferably about 80% cell confluency is used
as the
end point for each passage to maintain cultures with optimal cell numbers and
viability.
ISOLATION OF DENTAL PULP STROMAL CELLS
Tooth structure can be divided into two parts anatomically, namely the crown
and the
root, separated by the cementoenamel junction (CEJ). The CEJ is the crown part
that
consists of enamel, dentin and pulp chamber. The anatomic root is located
below the
CEJ that is covered with cementum and also has a pulp canal in the root area.
The
development of teeth begins embryologically as a series of interactions
between the oral
epithelium and neural crest-derived ectomesenchymal cells of the early jaw.
Dental pulp consists of loose connective tissue that is confined within the
pulp chamber
and root canals of the tooth. The composition of a dental pulp is by weight
75% water
and 25% organic material comprised of a plentiful matrix of cells. Cells such
as
odontoblasts, fibroblasts, undifferentiated ectomesencyhmal cells, macrophages
and
other immunocompetent cells are the principal fibres that can be found within
the dental
pulp. Cells within the pulp provide the mature pulp with odontogenic,
nutritive, sensory
and defensive functions that allow for the preservation of vitality during
normal
homeostatic maintenance and post injury repair and regeneration. Odontoblasts
are
highly specialized cells that are responsible for the secretion and
mineralization of the
fibrillar extracellular matrix of dentin. Fibroblasts are important in
formation and
maintenance of the pulp matrix consistency of collagen. Undifferentiated
ectomesenchymal cells are found throughout the pulp core which is therefore a
source
of material for stromal cell applications.
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Extraction of dental pulp from tooth
An intra-oral examination is done to assess the pulp status of the subjects.
Samples are
collected from the pulp tissue that is extirpated from freshly extracted
deciduous teeth.
The extracted deciduous tooth is kept in a specimen container filled with
normal saline
to maintain the moisture content within the tooth. The cutting of the tooth
and
extirpation of the dental pulp tissue should commence as soon as possible
after tooth
extraction from the subject. Extracted teeth are sterilized externally with
Povidone
iodine (PVP-I, Sigma Aldrich, St Louis, USA) and washed with distilled water
to
minimize the contaminants from the tooth surface contaminating the extirpated
dental
pulp tissue.
A tooth holder is used to hold the teeth securely, as illustrated in Figure 5.
The tooth
holder comprises a modified circular rotating drill chuck 2 with three jaws 4
fixed to a
stainless steel base 6. The drill chuck includes a central channel 8 through
which water
may be pumped via tube 10 to deploy saline or distilled water to the tooth and
for
cleaning the device. The jaws 10 may be extended or retracted via rotation of
the collar
12, which also alters the aperture size at the tips of the jaws to allow a
tooth 14 to be
gripped firmly. The jaws may be provided with grooves 16 or steps to allow for
precise
measurement and placement of the tooth to optimize dental pulp extirpation.
Water may
be also dripped from a tube 18 located above the chuck 2.
The tooth is sectioned horizontally at the cementoenamel junction using a
sterilized
diamond disc straight hand piece to expose the pulp. Extirpation of the dental
pulp
tissue is done under aseptic conditions. Copius irrigation with saline or
distilled water is
provided throughout the procedure which acts as a lubricant during and
throughout the
process, and also serves to reduce the amount of heat generated from the
cutting
process. This minimizes the destruction of tissue from heat. The dental pulp
tissue is
then transferred into a 1.5 ml tube containing DMEM Knock Out Basal Media
(Gibco,
Grand Island, NY). The extirpated pulp tissue sample is immediately sent to
the
laboratory for isolation process.
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Dental pulp stromal cell isolation process
Dental pulp tissues are processed in a Class II Biological Safety Cabinet
(BSC) with the
following environment conditions; room temperature (25 C 5), relative humidity
(65% 10). The following process is carried out entirely in a BSC sterilized
with 70%
ethanol and 30 minutes UV radiation. The 1.5m1 microcentrifuge tubes
containing
dental pulp tissues are wiped with 70% ethanol. The dental pulp tissues are
washed
three times in a washing buffer solution that contains Dulbecco's Phosphate-
Buffered
Saline without calcium and magnesium (DPBS -Ca', -Mg', Invitrogen, USA), 0.85%
of penicillin-streptomycin and 0.85% of antibiotic-antimycotic. Then, the
tissue is
minced into small fragments prior to Collagenase Type I treatment (Gibco,
Grand
Island, NY). After that, the tissue is incubated at 37 C for 30 minutes. Next,
the tissue is
transferred into the 15 ml tube (BD Bioscience, Franklin Lakes, NJ, USA)
containing 8
ml of MSC culture media. The tissue is centrifuged for 6 minutes at 1250 rpm.
The
supernatant is discarded and the pellet is gently resuspended with 10 ml of
MSC culture
media. Then, the cells are seeded in a T25 culture flask together with the
growth media
and incubated in a 5% CO2 incubator humidified with 95% of air or using a low
oxygen
hypoxia incubator. The medium is replaced every three days and monitored for
any
signs of contamination. After the primary culture becomes confluent in
approximately
10-20 days, cells are collected by trypsinization using 0.05% Trypsin-EDTA
(Gibco,
invitrogen) and processed for subsequent subcultures.
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ISOLATION OF FORESKIN STROMAL CELLS
Transporting the foreskin upon circumcision
Foreskin fibroblast-like stromal cells (FDSCs) are progenitors isolated from
human
5 tissue that can differentiate into many cell types. These cells are
mainly found on the
dermis of the foreskin. Human foreskin is obtained from neonates, children,
teenagers
or adults of male sex during circumcision and placed into sterile 50m1
polypropylene
conical centrifuge tubes pre-added with 25m1 of transport buffer stored at 4
C, 5 C, 6 C,
7 C, 8 C, 9 C, 10 C. This entire process should be done in a room with
temperature
10 ranging from 20 C to 25 C and the foreskin must be placed into the
transport buffer
solution within 24 hours to maintain tissue viability. The foreskin is then
transported to
the laboratory for mesenchymal stromal cell isolation.
Foreskin cleaning process
15 Foreskin tissues are processed in a Class II Biological Safety Cabinet
(BSC) with the
following environment conditions; room temperature (25 C 5), relative humidity
(65% 10). The following process is carried out entirely in a BSC sterilized
with 70%
ethanol and 30 minutes UV radiation. The 50m1 polypropylene conical centrifuge
tubes
containing the foreskin tissues are wiped with 70% ethanol. The sample is then
transferred into a 50m1 polypropylene conical centrifuge tubes pre-added with
30m1
SDW. The sample is washed by inverting the tubes repeatedly to remove traces
of blood
and to prevent any contamination. This step is repeated at least three times
to ensure the
foreskin is thoroughly washed using fresh SDW in a 50m1 polypropylene conical
centrifuge tube. The foreskin is then immersed in 70% ethanol for 25 to 30
seconds to
sterilize the surface and immediately immersed into SDW for another 25 to 30
seconds
to remove ethanol residues and create an immediate hypotonic environment on
the
skin's surface to eliminate microbes. The tissue is then transferred into a
50m1
polypropylene conical centrifuge tubes pre-added with 25m1 10% v/v Antibiotic-
Antimycotic and 10% v/v Penicillin-Streptomycin in PBS. The tubes are
incubated at
around 28-38 C, about 4-6% CO2 for around 2-16 hours, preferably 3TC, 5% CO2
for 2
hours/overnight. The incubator is programmed at 3TC, 5% CO2 to provide an
optimal
environment for the cord tissues/cells to maintain viability.
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Foreskin fibroblast-like stromal cell (FDSC) isolation process
Prior to incubation, the sample is washed with PBS twice for 30 to 60 seconds,
to
remove traces of Antibiotic-Antimycotic and Penicillin-Streptomycin. The
foreskin is
then cut into small pieces (2-5mm) and transferred to a fresh 50m1
polypropylene
conical centrifuge tubes containing 20m1 of 3% w/v collagenase type IV
dissolved in
DMEM-KO. The sample is allowed to be partially digested at around 28-38 C,
about 4-
6% CO2, preferably 37 C, 5% CO2, for 4 to 10 hours. 400 1 of 0.25% Trypsin-
EDTA is
added into the partially digested sample and incubated at 37 C, 5% CO2 for 5
minutes to
detach cells from the tissue. To neutralize the enzymatic action, 20m1 of MSC
culture
media is added into the tube and centrifuged at about 1500-2500rpm, 700-1000g
force/RCF, for around 8-12minutes, at about 25-28 C, preferably about 1800rpm
for 10
minutes at 25 C. It is crucial to neutralize the enzymatic action as cell
viability will be
reduced if Trypsin remains in contact with the cells for too long (typically
it should not
exceed 15 minutes). Prior to centrifugation, 50% of the supernatant is
carefully removed
by pipetting to eliminate excess MSC culture media and Trypsin. Undigested
semi-
transparent foreskin tissue fragments from the tube are transferred using
sterile tissue
forceps into a 60mm cell culture plate. This process is repeated until all
foreskin
fragments are transferred and left to air dry for 15minutes in the BSC. This
step is to
ensure the tissues adhere to the culture plate surface. Upon adherence of the
tissue
fragments onto the culture plate, 5-7m1 of MSC culture media are added gently
without
disrupting the attached tissues. The MSC culture media provides sufficient
nutrients and
optimal environment for the survival of the tissues. Culture plates are then
transferred to
a CO2 incubator or low oxygen hypoxia incubator and incubated for 24 hours.
After 24
hours incubation, 2/3 of the media is pipetted out gently without disrupting
adhered
tissues and replaced with fresh MSC culture media. The culture plates are
transferred
back to the incubator and an additional 5m1 of fresh MSC culture media is
added in the
next 24 hours for continuous supply of nutrients for cell growth and also to
remove dead
or apoptotic cells from the culture. The culture is observed using an inverted
microscope
every day and 2/3 of media is replaced every 2 to 4 days until the cells reach
80%
confluency until subcultured to passage 1 stock. 80% cell confluency is used
as the end
point for each passage to maintain cultures with optimal cell numbers and
viability.
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ISOLATION OF CORNEAL LIMBAL EPITHELIAL STROMAL CELLS
Corneal limbal epithelial stromal cells (CLSCs) is a non-keratinized
epithelium
composed of a cluster of cells with a capacity for self-renewal. These cells
are localized
at the basal cell layer of the limbus which is responsible for the
regeneration of corneal
epithelium and to maintain the cornea. CLSCs differ from the corneal
epithelial cells
due to the lack of COM eo-specific differentiation kera tins (K 3/K 1 2)
expression,
connexin 43 -in edi ated gap junction intercellular communication, p63 nuclear
transcription factor, cell cycle duration, and label retaining properties.
Transporting corneal tissue upon keratoplasty or from any other procedure
Human corneal tissue, obtained via any form of keratoplasty surgery done for
patients
with keratoconus, corneal scarring, Fuchs' dystrophy, Lattice dystrophy and
other
cornea related diseases which requires cornea transplantation, or human
corneal tissue
obtained in any other manner, is placed into a sterile 50m! polypropylene
conical
centrifuge tubes pre-added with 25m! of transport buffer stored at 4 C, 5 C, 6
C, 7 C,
8 C, 9 C, 10 C. This entire process should be done in a room with temperature
ranging
from 20 C to 25 C and the corneal tissue must be placed into the transport
buffer
within 24 hours of the procedure to maintain tissue viability. The cornea
tissue is then
transported to the laboratory for stromal cell isolation.
Corneal tissue cleaning process
Corneal tissues is processed in a Class II Biological Safety Cabinet (BSC)
with the
following environment conditions; room temperature (25 C 5), relative humidity
(65% 10). The following is carried out entirely in a BSC sterilized with 70%
ethanol
and 30 minutes UV radiation. The 50m! polypropylene conical centrifuge tubes
containing the cornea tissues are wiped with 70% ethanol. The sample is then
transferred into a 50m! polypropylene conical centrifuge tube pre-added with
30m!
SDW. The sample is washed by inverting the tubes repeatedly to prevent
contamination.
This step is repeated at least three times to ensure the tissue is thoroughly
washed using
fresh SDW and 50m! polypropylene conical centrifuge tubes. The tissue is
transferred
into a 50m! polypropylene conical centrifuge tube pre-added with 25m! 10% v/v
Antibiotic-Antimycotic and 10% v/v Penicillin-Streptomycin in PBS. The tube is
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incubated at 37 C, 5% CO2 for 2 hours/overnight. The incubator is programmed
at 37 C,
5% CO2 to provide an optimal environment for the cord tissues/cells to
maintain its
viability.
Corneal limbal epithelium stromal cell (CLSC) isolation process
Prior to incubation, the sample is washed with PBS twice for 30 to 60 seconds,
to
remove traces of Antibiotic-Antimycotic and Penicillin-Streptomycin. The
tissue is then
cut into small pieces (2-5mm) and transferred to a fresh 50m1 polypropylene
conical
centrifuge tubes containing 20m1 of 3% w/v collagenase type IV dissolved in
DMEM-
KO. The sample is allowed to be partially digested at around 28-38 C, about 4-
6% CO2,
preferably 37 C, 5% CO2, for 2 to 4 hours. 400 1 of 0.25% Trypsin-EDTA is
added into
the partially digested sample and incubated at around 28-38 C, about 4-6% CO2,
preferably 37 C, 5% CO2, for 5 minutes to detach cells from the tissue. To
neutralize the
enzymatic action, 20m1 of MSC culture media is added into the tube and
centrifuged at
about 1500-2500rpm, 700-1000g force/RCF, for around 8-12minutes, at about 25-
28 C,
preferably about 1800rpm for 10 minutes at 25 C. It is crucial to neutralize
the
enzymatic action as cell viability will be reduced if Trypsin remains in
contact with the
cells for too long. Prior to centrifugation, 50% of the supernatant is
carefully removed
by pipetting to eliminate excess MSC culture media and Trypsin. Undigested
tissue
fragments and the remaining MSC culture media from the tube are aspirated into
60mm
cell culture plate. An additional 5-7m1 of MSC culture media is added into the
culture
plate. The MSC culture media provides sufficient nutrients and optimal
environment for
the survival of the tissues. The culture plates are then transferred to a CO2
incubator or a
low oxygen hypoxia incubator and incubated for 24 hours. After 24 hours
incubation,
2/3 of the media is pipetted out gently without disrupting adhered tissues and
replaced
with fresh MSC culture media. The culture plates are transferred back to the
CO2
incubator and an additional 5m1 of fresh MSC culture media is added in the
next 24
hours for continuous supply of nutrients for cell growth and also to remove
dead or
apoptotic cells from the culture. The culture is observed using an inverted
microscope
every day and 2/3 of media is replaced every 2 to 4 days until the cells reach
80%
confluency until subcultured to passage 1 stock. 80% cell confluency is used
as the end
point for each passage to maintain cultures with optimal cell numbers and
viability.
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EXPANSION OF MESENCHYMAL STROMAL CELLS (MSCs)
Expansion of MSCs from passage 0 to passage 1 stock.
Prior to subculture from passage 0 to passage 1 of MSCs derived from umbilical
cord,
dental pulp, foreskin and cornea, a Class II BSC with following environment
conditions; room temperature (25 C 5), relative humidity (65% 10), is
sterilized with
70% ethanol and 30 minutes UV radiation. The 60mm cell culture plates with 80%
confluence cells are transferred from the CO2 incubator to the BSC. Culture
media is
pipetted out completely and the culture plate is washed with 10m1 PBS (without
Magnesium and Calcium ions) twice. Each culture plate is gently swirled 10 to
15 times
during washing process and the remaining PBS is pipetted out. 2m1 of pre
warmed
TrypLETm Express or Accutase or Trypsin-EDTA is added into each culture plates
and
incubated at 37 C, 5% CO2 until the cells detach. Cell culture monolayers are
observed
under an inverted microscope for cell detachment from the surface of the
culture plate
and the culture plate is tapped gently to dislodge cells. 5m1 of pre-warmed
MSC culture
media is added to the culture plate and swirled gently. MSCs suspension from
culture
plates are transferred through pipetting into 15m1 polypropylene conical
centrifuge
tubes each. The tubes are then centrifuged at 1200rpm for 6 minutes at 25 C.
The
supernatant is discarded and cell pellets are re-suspended with 5m1 of pre-
warmed MSC
culture media. After performing the cell count either by haemocytometer with
4%
Trypan blue exclusion method or by using automated cell counter, MSCs cells
are
seeded into T175 culture flask at a 1500ce11s/cm2 seeding density with the
addition of
15m1 of MSC culture media and the flasks are moved forward and back gently to
cause
the cells to spread evenly. The remaining cells at passage 0 are then
cryopreserved as
MSCs SCO. The flasks containing MSCs SC1, are then incubated at 37 C in 5%
humidified CO2 incubator or low oxygen hypoxia incubator. Every 3 days cells
are
observed under inverted microscope and 8m1 of MSC culture media are replaced
until
cells reach 80% confluency. Upon reaching 80% confluency, WJSC SC1 cell
cultures
are sub-cultured into passage 2.
Expansion of MSCs from passage 1 to passage 2 stock.
Prior to subculture MSCs from passage 1 to passage 2 a Class II BSC with
following
environment conditions; room temperature (25 C 5), relative humidity (65% 10)
are
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sterilized with 70% ethanol and 30 minutes UV radiation. The T175 flasks with
80%
confluence cells are transferred from the CO2 incubator to the BSC. Spent
media is
aspirated out completely and washed with 20m1 PBS (without Magnesium and
Calcium
ions) twice. Each of the flasks is gently swirled several times during washing
process
5 and remaining PBS is aspirated out. 5m1 of pre warmed TrypLETm Express is
added into
each flasks and incubated at 37 C, 5% CO2 until cells detach. Cell culture
monolayers
are observed under an inverted microscope for cell detachment from the surface
of the
flask and the flask is tapped gently to dislodge cells. 15m1 of pre-warmed MSC
culture
media is added to the flask and swirled gently. MSCs suspension from T175
flasks is
10 transferred through pipetting into 50m1 polypropylene conical centrifuge
tubes each.
The tubes are centrifuged at about 1000-1250rpm, for around 5-7 minutes, at
about 25-
28 C, preferably about 1200rpm for 6 minutes at 25 C. The supernatant is
discarded and
cell pellets are re-suspended with 5m1 of pre-warmed MSC culture media. After
performing a cell count, MSCs are seeded into T175 culture flask at a
1500ce11s/cm2
15 seeding density for optimal cell proliferation while reducing the
chances of inducing
biological stress into the cell population. 15m1 of MSC culture media are then
added the
flasks are moved forward and back gently to cause and ensure that the cells
spread
evenly. Remaining cells at passage 1 are cryopreserved as MSCs SC1. The flasks
containing MSCs 5C2, are then incubated at 37 C in a 5% humidified CO2
incubator or
20 with a low oxygen hypoxia incubator. The cells are observed under
inverted microscope
every 2 to 4 days and 8m1 of MSC culture media is replaced during each
observation.
This process is repeated until cells reach 80% confluency. Upon reaching 80%
confluency, MSCs 5C2 cell cultures are sub-cultured into passage 3 as a
finished
product.
Expansion of MSCs from passage 2 to passage 3 as finished product.
Prior to subculture MSCs from passage 2 to passage 3 a Class II BSC with
following
environment conditions; room temperature (25 C 5), relative humidity (65% 10)
is
sterilized with 70% ethanol and 30 minutes UV radiation. The T175 flasks with
80%
confluence cells are transferred from CO2 incubator to the BSC. Spent media is
aspirated out completely and the T175 flask is washed with 20m1 PBS (without
Magnesium and Calcium ions) twice. Each of the flasks are gently swirled
several times
during washing process and remaining PBS is aspirated out. 5m1 of pre warmed
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TrypLETm Express is added into each flask and incubated at around 28-38 C,
about 4-
6% CO2, preferably 37 C, 5% CO2, until the cells detach (i.e. stop adhering to
the flask
wall). Cell culture monolayers are observed under an inverted microscope for
cell
detachment from the surface of the flask and the flask is tapped gently to
dislodge cells.
15m1 of pre-warmed MSC culture media is added to the flask and swirled gently.
MSCs
suspension from T175 flasks are transferred via pipetting into 50m1
polypropylene
conical centrifuge tubes each. The tubes are centrifuged at about 1000-
1250rpm, for
around 5-7 minutes, at about 25-28 C, preferably about 1200rpm for 6 minutes
at 25 C.
The supernatant is discarded and cell pellets are re-suspended with 5m1 of pre-
warmed
MSC culture media. Upon performing a cell count, cells at passage 3 (MSCs SC3)
are
prepared for cryopreservation.
CRYOPRESERVATION
Cryopreservation of MSCs passage 3 (MSCs SC3) as finished product.
Prior to cryopreservation of MSCs finished product at passage 3 a Class II BSC
with
following environment conditions; room temperature (25 C 5), relative humidity
(65% 10) is sterilized with 70% ethanol and 30 minutes UV radiation. Freezing
media
is used as cryopreservation media. The appropriate volume of freezing media is
prepared fresh according to the formula in Table 4 below:
Table 4
No. Freezing media component Concentration
1. MSC culture media 90%
2. Dimethyl sulfoxide (DMSO) 10%
Freezing media is not allowed to be exposed to light and is kept at 4 C 2 ¨
this is to
negate the effects of light on DMSO. Cryovials are sterilized using 70%
ethanol and
labelled using a permanent cryo marker pen. MSCs SC3 pellets in 50m1
polypropylene
conical centrifuge tubes are gently exposed to desired volume of cold Freezing
media
by using a micropipette. Cells are carefully re-suspended to avoid gas bubble
formation.
The desired volume of cell suspension is transferred into pre-labelled
cryovials.
Freezing density and required volume of freezing media are illustrated in
Table 5.
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Table 5
No. Freezing density (cells) Volume of Freezing media
(IA)
1. 5 x 106 800
2. 10 x 106 1000
3. 20 x 106 1200
Cryovials are immediately transferred to a -80 C freezer for 24 hours and
finally kept in
liquid nitrogen cryotank the following day. Cryovials are kept at -80 C
freezer to allow
for a gradual cooling process to occur (1 C/minute) till the vials reach -50 C
prior to the
vials being transferred into a liquid nitrogen cryotank. This is to reduce
intracellular
crystal formation thereby maintaining the optimal amount of viable cells.
Location and
details of each cryovial stored are recorded to ease the retrieval process.
OBSERVATIONS
Cell morphology
Cell morphology of WJSCs, SHED, FDSCs and CLSCs at 80 to 90% were observed at
passage 3 using an inverted microscope. As illustrated in Figure 1, the
adherent MSCs
displayed typical fibroblast-like spindle shaped morphology which confirm
established
descriptions of stromal-like cell morphology.
Immunophenotyping of MSC markers
Immunophenotyping was carried out via flow cytometry at SC3. Upon reaching 80%
confluency, the cells were harvested using 0.05% trypsin-EDTA (Invitrogen) and
resuspended in Dulbecco's Phosphate Buffer Saline (DPBS; Invitrogen) at a cell
density
of 1.5x106 cells/mL. Two hundred microliters of the cell suspension (3x105
cells) were
incubated with the labelled antibodies in the dark for 1 hour at 37 C.
Antibodies
.. conjugated to fluorochromes are susceptible to photobleaching and should be
protected
from light during all phases of an experiment. The following antibodies were
used to
mark the cell surface epitopes: CD90-phycoerythrin (PE), CD44-PE, CD73-PE,
CD166-
PE and CD34-PE, CD45-fluoroisothiocyanate (FITC), and HLA-DR-FITC (all from BD
Pharmingen). All analyses are standardized against negative control cells
incubated with
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isotype-specific immunoglobulin (Ig) Gl-PE and IgGl-FITC (BD Pharmingen). At
least 10,000 events are acquired on BD FACS Calibur flow cytometer to analyse
the
total gated cell population. Purity and MSCs-like phenotype of the isolated
cells were
confirmed through flow cytometry result, wherein more than 90% of WJSCs, SHED,
FDSCs and CLSCs expressed MSC markers (CD44, CD73, CD90) and >60% for
CD166. The cells also expressed less than 0.5% for hematopoietic markers
(CD34,
CD45 and HLA-DR). These results are illustrated in Table 6 below:
Table 6
WJSC SHED FDSC CLSC
CD90
CD44
CD73
CD166
CD34
CD45
HLA-DR
In vitro multilineage differentiation
The cultures of respective sources are initiated at a density of 1500
cells/cm2 in 6-well
plates and are grown to confluence and subjected to differentiation into
adipogenic,
chondrogenic, and osteogenic lineages. Adipogenic lineage is stimulated by
inducing
the cells with 10% FBS, 200 mmol/L indomethacin, 0.5 mmol/L 3-isobuty1-1-
methyxanthine ("BMX), 10 mg/mL insulin, and 1 mmol/L dexamethasone (all
reagents
from Sigma Aldrich). Lipid droplets are visualized by staining with oil red 0
staining
(Sigma Aldrich). For chondrogenesis differentiation, cells are cultured in
media
supplemented with ITS+1 (Sigma Aldrich), 50 mmol/L L-ascorbic acid-2
phosphates
(Sigma Aldrich), 55 mmol/L sodium pyruvate (Invitrogen), 25 mmol/L L-proline
(Sigma Aldrich), and 10 ng/mL of transforming growth factor-beta (TGF-b)
(Sigma
Aldrich). Assessment of proteoglycan accumulation is visualized by alcian blue
staining
(Sigma Adrich). The osteogenic differentiation is stimulated in a 3-week
culture in
media supplemented with 10% FBS, 100 mon dexamethasone, 10 mmol/L b-glycerol
phosphate (Fluka, Buchs, Switzerland), and 100 mmol/L of L-ascorbic acid-2
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phosphates. Assessment of calcium accumulation is visualized by von Kossa
staining.
WISCs, SHED, FDSCs and CLSCs were able to differentiate into chondrogenic,
adipogenic and osteogenic lineages in vitro). Chondrogenic differentiation
revealed
production of extracellular matrix glycosaminoglycans when stained with Alcian
blue
and differentiation of adipocyte were confirmed with oil red 0 staining for
presence of
lipid droplets vacuole. In osteocyte differentiation medium, spindle-shaped
dental
MSCs metamorphosed into cubical cells with visible mineralized foci detected
as black
spotted areas with von Kossa staining.
Differentiation of MSCs into dopaminergic-like cell
Embryoid Body (EB) formation
Stage 1, EB formation
At 80% confluency, cells are transferred into a non-adherent petri dish
containing a
neural-inducing medium (NIM) composed of 92% D-MEM/ F-12 (1:1), 6000 mg/L D-
glucose, 5% KnockOut SR, 2 mM L-glutamine, 1% MEM non-essential amino acids
solution, 0.1 mM b-mercaptoethanol, and 1% N2 supplement (all from Gibco BRL)
via
hanging drop method. The EBs are well developed under these conditions in
which cell
aggregates form within 24 hours of incubation.
Dopa media A
The EBs are aspirated to super-hydrophilic plates (Costar, Cambridge, MA) in
Neurobasal A media (GIBCO/Invitrogen, Carlsbad, CA) and supplemented with B27
(Invitrogen, Carlsbad, CA), 20 ng/mL basic fibroblast growth factor (bFGF;
Invitrogen)
and 20 ng/mL epidermal growth factor (EGF; Invitrogen). Fresh culture medium
including EGF and bFGF is added to the medium after 4-5 days of culture.
Dopa media B
The EBs were induced with a cocktail of 200 ng/mL sonic hedgehog (SHH), 100
ng/mL
.. fibroblast growth factor 8 (FGF8), 10 ng/mL glial cell line-derived
neurotrophic factor
(GDNF; R&D Systems, Minneapolis, MN), and 10 [tM forskolin (PeproTech EC,
London, England). Cells are then cultured at 37 C with 5% CO 2 for 7 days.
Half of the
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medium is replaced every 2-3 days. After 7 days of cultivation, the cells
differentiate
into neuronal cells and are tested for neural cell marker expression.
As illustrated in Figure 2, MSCs derived from WJSCs, SHED, FDSCs and CLSCs are
5 able to form cell aggregates/neurospheres in NIM media. However neurite
outgrowth
was only observed in SHED, FDSCs and CLSCs aggregates/neurospheres.
Quantitative reverse-transcription Polymerase Chain Reaction
The cell pellets from respective sources are collected and total RNA is
isolated using
10 TRIZOL method (Invitrogen), as per the manufacturer's protocol. The RNA is
then
quantified using a spectrophotometer (Agilent, NanoDrop, Technologies Inc.)
and the
purity is assessed by the absorbance value at ratio of 260:280 nm. The RNA is
stored at
-80 C. The first-strand complementary DNA (cDNA) is synthesized using 1 mg RNA
treated with RNase-OUT ribonuclease inhibitor and the Superscript II First
Strand
15 Synthesis system (Invitrogen) according to the manufacturer's
instructions. PCR is
performed in 0.2 mL Eppendorf tubes (Axygen) with a final volume to 12.5 mL.
cDNA
amplification is performed in a thermocycler using Taq polymerase supplied
with KC1
buffer and 1.5mM MgCl2 (Invitrogen) at 94 C for 1 min, 58 C for 30 s and 72 C
for 1
min. PCR products are resolved on 1.5% agarose (Invitrogen) gel run in lx Tris
borate-
20 EDTA buffer. The primer sequences are listed in Table 7. As illustrated
in Figure 3, the
expressions of some genes in the semi-quantitative RT¨PCR analysis are
quantified in
duplicate, using SYBR Green Master Mix (Applied Biosystems). PCR reactions are
performed on an ABI 7900HT RT¨PCR system (Applied Biosystems) and SDS v 2.1
software was used to analyse the results and to prove the immunophenotyping
25 characteristics. All measurements are normalized to 18s rRNA.
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Table 7:
Gene SEQ ID Primers
No.
Nestin 1 Forward: 5'-CAGCGTTGGAACAGAGGTTGG-3'
2 Reverse: 5'- TGGCACAGGTGTCTCAAGGGTAG-3'
8-Tubulin III 3 Forward: 5'-CAACAGCACGGCCATCCAGG-3'
4 Reverse: 5- CTTGGGGCCCTGGGCCTCCGA-3'
GAD 65 5 Forward: 5'- ATGACACTGGAGACAAGGCC-3'
6 Reverse: 5'- TTGGTAGCTGACCATTGTGG-3'
GAD67 7 Forward: 5'- TGGAAGTGGTGGACATACTCC-3'
8 Reverse: 5.- AAGTACTTGTAGCGAGCAGCC-3'
Tyrosine hydroxylase (TH) 9 Forward: 5'-GGTT000AAGAAAAGTGTCAG-3'
Reverse: 5'-GGTGTAGACCTCCTTCCAG-3'
Immunocytochemistry analysis of the MSCs
Cells from respective sources are fixed for 20 min in 4% ice cold
paraformaldehyde,
5 treated with 0.1% Triton-X for optimal penetration of cell membranes, and
incubated at
room temperature (RT) in a blocking solution (0.5% BSA; Sigma Aldrich) for 30
min.
Primary antibodies [Nestin (mouse, Abcam), fl-tubulin (mouse, Abcam), GAD65/67
(rabbit, Abcam), TH (Rabbit, Abcam) with dilution of 1:400] are incubated
overnight at
4 C, washed with DPBS, and then incubated with secondary antibodies (either
10 .. fluorescein isothiocyanate [FITC]-conjugated IgG or rhodamine-conjugated
IgG) at RT
for 90 min. Slides were counterstained with 4',6'-diamidino-2-phenylindole
dihydrochloride (DAPI, Chemicon, Temecula, CA, USA) for 5 min. Fluorescent
images
were captured using Nikon-Eclipse-90i microscope (Nikon, Tokyo, Japan,
http://www.nikon.com).
As illustrated in figure 4, the immunocytochemistry results show that WJSCs,
SHED,
FDSCs and CLSCs express Tyrosine hydroxylase (TH), fl-tubulin, Nestin and
Glutamate decarboxylase 65 and 65 (GAD65/67) protein post dopaminergic neuron-
like
differentiation. However, in undifferentiated SHED, expression of Nestin and
GAD65/67 is observed while expression of 13-tubulin, GAD65/67 and TH is
observed in
undifferentiated WJSCs. This finding shows that SHED and WJSCs are the best
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candidates for any therapeutic application for dopaminergic neuronal
degenerative
disease.
Cells obtained from the protocol described herein are able to retain 83% to
89% of
viability (non-apoptopic) at passage 3. Prior art percentages achieved at
passage 3 are
around 70%-80% viability using 4% trypan blue exclusion method. However, usage
of
the 4% trypan blue exclusion method includes cell in late and early apoptosis
as being
viable, and as such, is not an accurate means of counting for cell viability.
In this
protocol the method used is more accurate, namely the Annexin V apoptosis
detection
method by flow cytometry, to find out the exact percentage of non-apoptotic
cells,
excluding early and late apoptotic cells.
It will be appreciated by persons skilled in the art that the present
invention may also
include further additional modifications made to the system which does not
affect the
overall functioning of the system.
