Download Rabbit Adipose-Derived Mesenchymal Stem Cells

User Manual
OriCellTM Rabbit Adipose-Derived
Mesenchymal Stem Cells (ADSCs)
Cat. No. RBXMD-01001
Table of Contents
Contents and Storage ………………………………………………………………………………………3
Product Introduction…………………………………………………………………………………………3
Cell Characteristics and Identity …………………………………………………………………………3
Product Applications ………………………………………………………………………………………4
General Handling Principles ………………………………………………………………………………4
Culturing OriCellTM Rabbit ADSCs
Thawing and Establishing OriCellTM Rabbit ADSCs …………………………………………………4
Passaging Cyagen OriCellTM Rabbit ADSCs ………………………………….……………………… 6
Differentiation of OriCellTM Rabbit ADSCs ……………………………………………………………8
Cryopreservation of OriCellTM Rabbit ADSCs ………………..………………………………………12
Appendix … ……………… ……………………………………………………………14
Troubleshooting ……………………………………………………………………………………………14
Related Products …………………………………………………………………………………………15
References ……………………………………………………………………………………………………15
CONTENTS AND STORAGE
Product Name
Rabbit Adipose‐Derived Mesenchymal
Stem Cells
Catalog No.
RBXMD‐01001
Amount per Vial
1×106 Cells
Cryopreserved At
Second Passage
Storage Condition
Liquid Nitrogen
CAUTION: Please handle this product as a potentially biohazardous material. This
product contains Dimethyl Sulfoxide (DMSO), a hazardous material, in the freezing
medium.
PRODUCT INTRODUCTION
Adipose-Derived Mesenchymal Stem Cells (ADSCs) are multipotent stem cells that can
differentiate into a variety of cell types, including osteocytes, adipocytes and
chondrocytes. They have wide application potentials in tissue engineering, cell therapy
and gene therapy.
OriCellTM Rabbit Adipose-derived Mesenchymal Stem Cells (ADSCs) are derived from
adipose tissue of healthy Rabbit by liposuction. They have a strong capacity for selfrenewal while maintaining multipotency.
In addition, these cells have been tested for:
•
Exogenous Factors: bacterial/fungal contamination, mycoplasma contamination,
and endotoxin contamination.
•
Characteristics: post-thaw viability, cell cycle, verification of undifferentiated
state, and differentiation potential.
This product is intended for laboratory research use only. It is not intended for
diagnostic, therapeutic, clinical, household, or any other applications.
CELL CHARACTERISTICS AND IDENTITY
•
Strong capacity to expand. Can be passaged at least 5 times.
•
Multipotent differentiation ability along the osteogenic, chondrogenic, and
IMPI0022A2 RBXMD‐01001
Page 3 of 15
adipo
ogenic linea
ages.
PRODUC
CT APPL
LICATIO
ONS
Adip
pose-Derive
ed Mesench
hymal Stem
m Cells (ADSCs) have become a popular res
search
targ
get due to their
t
potenttial use in rregenerativ
ve medicine
e and tissue
e engineering (in
area
as such as cardiovascular, neura l and ortho
opaedic dise
ease).
CellTM Rabbit Adipose-Derived Me
esenchymal Stem Cells can be ussed as cell models to
OriC
testt and evaluate the imm
munoreacti ons, prolife
eration, imm
migration a
and differen
ntiation of
ADS
SCs both in vivo and in
n vitro.
GENERA
AL HAND
DLING PRINCIP
P
PLES
1.. Aseptic handling
h
of the producct is necess
sary through
hout.
2.. Once the
e cells have been estab
blished, alw
ways freeze
e up severa
al vials of AdiposeDerived Mesenchym
M
mal Stem Ce
ells (ADSCs
s) as a backup.
Note: The O
OriCellTM RABBIT ADSC
Cs can be fr
rozen/thaw
wed at leastt one times
s.
N
nance of ce
ells, we reco
ommend th
he seeding density to be 2.03.. For general mainten
2
c
.
2.5×104cells/cm
4.. For all studies, it is strongly re
ecommende
ed to use ce
ells that are
e at, or und
der, an
p
number of 10
0.
original passage
5.. For general mainten
nance of ce
ells, we reco
ommend th
hat the med
dium is cha
anged if it
e pH indicattor in culture medium appears ye
ellow). In general,
g
becomes acidic (the
very three days.
d
change the growth medium ev
et Rabbit AD
DSCs overg
grow, as it will
w result in contact in
nhibition. When
W
the
6.. Do not le
cells are 80-90% co
onfluent, su
ubculturing the cells is
s strongly rrecommend
ded.
Note: We sttrongly reco
ommend th
he use of Or
riCellTM cult
ture media and other related
N
re
eagents for optimal re
esults.
THAWIN
T
NG AND ESTABL
LISHING
G OriCelllTM RABBIT ADS
SCs
M
Materials Required
R
ƒ
OriCellTM Rabbit Adipose-Deriv
ved Stem Cells (Cat. No.
N RBXMD--01001)
ƒ
OriCellTM Adipose-De
erived Stem
m Cell Grow
wth Medium
m (Cat. No. GUXMD-90
0011)
Th
hawing and
a
Estab
blishing R
Rabbit ADSCs
IMPI0022A2 RB
BXMD‐01001
Page 4 of 15
1.
Pre-warm
m the fully supplemen
s
ted (complete) OriCelllTM ADSCs Growth Me
edium to
37°C.
2.
L of OriCellTM ADSC G rowth Mediium to a 15
5 mL conica
al tube.
Add 9 mL
3.
Remove the cryovia
al of OriCelllTM Rabbit ADSCs
m liquid nitrrogen.
from
A
4.
t
the cryovial in a 37°C water bath until the last icce crystal disappears.
Quickly thaw
For optim
mal results, be sure to
o finish the thawing prrocedure wiithin 3 minutes. Be
careful not to subm
merge the en
ntire vial. Maximum cell
c viability
y is depend
dent on the
d complete thawing off frozen cells.
rapid and
ess than op
ptimal if the
e cells are thawed
t
forr more than
n 3 minutes
s.
N
Note: Resultts will be le
5.
As soon as
a the cells
s are complletely thawed, disinfec
ct the outsiide of the cryovial
c
with 70%
% v/v ethan
nol.
6.
TM
Use a pip
pette to transfer the ccells to the 15 mL coniical tube co
ontaining OriCell
O
Rabbit AD
DSC Growtth Medium inside a bio
osafety cabinet. Be ca
areful not to
t introduce
e
any bubb
bles during the transfe
er process.
7.
Rinse the
e vial with 1 mL of the
e medium to
t reduce cell loss. Su
ubsequently
y transfer
this 1 mL
L of cell sus
spension in
nto the conical tube.
8.
Gently mix
m the cell suspension
n by slowly pipetting up
u and dow
wn. Be care
eful not to
introduce
e any bubbles.
9.
Centrifug
ge the cell suspension
s
at 250 x g for 5 minu
utes.
0. Carefully
y aspirate off as much of the supernatant as
s possible a
and add 2-3
3 mL of
10
fresh OriCellTM Rabb
bit ADSC Grrowth Mediium (pre-warmed to 3
37°C).
11
1. Gently re
esuspend th
he cells in O
OriCellTM Ra
abbit ADSC
C Growth Me
edium.
12
2. Seed the
e cells into a T25 flask
k and add a sufficient amount
a
of OriCellTM Rabbit ADSC
C
Growth Medium.
M
Gently rock the culture
e flask to ev
venly distrib
bute the ce
ells.
13
3. Incubate
e the flask at
a 37°C ins ide a 5% CO
C 2 humidiffied incubattor.
14
4. The nextt day, chang
ge the med
dium with fresh
f
growth medium (pre-warmed to 37°C
C).
15
5. Change the
t
growth medium ev
very two da
ays until the cells are 80% confluent
thereafte
er.
16
6. When the
e cells are approximattely 80-90%
% confluent, they can
n be dissociated with
0.25%Trrypsin-0.04%EDTA an d passaged
d.
IMPI0022A2 RB
BXMD‐01001
Page 5 of 15
N
Note: Chang
ging Mediium
1.. Warm an
n appropriatte amount of medium to 37°C in a sterile co
ontainer. Replace
R
the
e
spent me
edium with the pre-wa
armed, fres
sh medium.. Once com
mpleted, retturn the
flask to the
t
incubator.
2.. Avoid rep
peated war
rming and c
cooling of the medium
m. If the en
ntire conten
nt is not
needed fo
or a single procedure,, transfer only
o
the req
quired volu me to a ste
erile
secondarry container.
TM
M
F
Fig.
1 OriCell Rabbit Adipo
ose-Derived Mesenchymal
M
Stem
S
Cells are
e established
PASSAG
GING OrriCellTM RABBIT
T ADSCs
M
Materials Required
R
•
0.25%Tryp
psin-0.04%EDTA (Cat. N
No. TEDTA-10001)
•
Phosphatte-Buffered
d Saline (1×
×PBS) (Cat. No. PBS-1
10001)
•
OriCellTM Rabbit Adipose-derive
ed Stem Ce
ells (Cat. No. RBXMD--01001)
•
OriCellTM Mesenchym
mal Stem C
Cell Growth Medium (C
Cat. No. GU
UXMX-9001
11)
a for Rabbitt adipose-d erived mes
senchymal stem cells ffrom other
r companies
N
Note: Media
s
ca
an be applie
ed to the cu
ulturing of OriCellTM Rabbit
R
Adipo
ose-derived
d Stem Cellls.
1.. Pre-warm
m the OriCe
ellTM MSC G rowth Medium, 1×PBS
S, and 0.25
5%Trypsin-0.04%ED
DTA solution
n to 37°C.
2.. Carefully aspirate th
he spent m edium from
m the 80-90
0% conflue
ent monolay
yer of
ADSCs.
3.. Add 1×PBS (6 mL fo
or T75 flask
k, 3 mL forr T25 flask). Be carefu
ul not to disturb the
er. Gently rock the fla
ask back an
nd forth to rinse the m
monolayer.
monolaye
4.. Aspirate 1×PBS off and discard
d.
5.. Repeat stteps 3-4 tw
wo or three times.
6.. Add 0.25
5%Trypsin-0.04%EDTA
A solution (2-3 mL for T75 flask,, 1 mL for T25
T
flask).
Gently ro
ock the flask back and
d forth to en
nsure that the entire m
monolayer is covered
with the 0.25%Tryp
psin-0.04%
%EDTA soluttion. Allow trypsinizatiion to continue until
IMPI0022A2 RB
BXMD‐01001
Page 6 of 15
oint, gently
the majo
ority of the cells (appro
oximately 80%)
8
are rounded up.. At this po
tap the side
s
of the flask
f
to rele
ease the majority of cells from th
he culture flask
f
surface.
xposed to th
he trypsin longer
l
than
n necessary
y (no
mportant: Avoid leaviing cells ex
Im
mo
ore than tw
wo minutes if using Cy
yagen’s tryp
psin-EDTA solution). C
Care should
d also
be
e taken thatt the cells are
a not forc
ced to detach prematu
urely as thiis may resu
ult in
clu
umping.
7.. After the
e cells are visibly
v
detacched, imme
ediately add the pre-w
warmed OriiCellTM MSC
C
Growth Medium
M
(6 mL for T75
5 flask, 3 mL
m for T25 flask) to ne utralize the
e
trypsiniza
ation.
8.. Gently piipette the medium
m
ove
er the cells
s to dislodge and resusspend the cells.
c
Repeat 5-6
5 times until all the ccells are dissociated from the fla
ask and eve
enly
dispersed
d into a single cell susspension.
9.. Transfer the dissociated cells i nto a 15 mL
m conical tu
ube.
10
0. Centrifug
ge at 250 x g for 5 mi nutes.
11
1. Carefully
y aspirate off as much of the supernatant as
s possible.
12
2. Add 2 mL
L of OriCellTM ADSC G rowth Mediium to the conical tub
be and genttly
resuspen
nd the cells thoroughly
y.
13
3. Plate the
e cells into appropriate
a
e flasks. OriCellTM Rab
bbit ADSCs can be split at 1:3 orr
other app
propriate ra
atios.
14
4. Add an appropriate
a
amount off medium to
o the cells. Incubate tthe cells at 37°C
inside a 5%
5 CO2 hu
umidified in
ncubator.
N
Note: Care should be taken
t
to av
void introdu
ucing bubbles during pipetting.
Ad
dditional Tips
Tiime to Cha
ange Mediium
It is recomm
mended to change
c
the culture me
edium if the
ere are too
o many dea
ad cells
affter passaging.
It is recomm
mended to change
c
the culture me
edium whenever the m
medium be
ecomes
ac
cidic, even if the cells do not rea
ach 80-90%
% confluenc
cy. The pH indicator in
n the
cu
ulture mediium will appear yellow
w when acid
dic. In gene
eral, chang
ge the grow
wth medium
m
ev
very three days.
d
bculture
Tiime to Sub
W
When OriCelllTM Rabbit ADSCs
A
are 80-90% confluent,
c
it is recomm
mended that the cells
s
be
e subculturred. Do not let the cellls overgrow
w as it will result
r
in co ntact inhibition.
IMPI0022A2 RB
BXMD‐01001
Page 7 of 15
Pas
ssage 3-40x
Pa
assage 3-100x
x
TM
Fig. 2 Images of OriCell
O
Rabbitt Adipose-Derrived Mesenchymal Stem Ce
ells at passage 3.
C DIFFE RENTIA
ATION USING
U
O
OriCellTM
OriCellTTM RABBIT ADSC
DIFFER
RENTIAT
TION ME
EDIA
OriCellTM Rab
bbit ADSCs can differe
entiate into a variety of
o cell typess including osteocytes
s,
dipocytes, and
a
chondrrocytes.
ad
O
Osteogenic Differen
ntiation
Materials Required
R
M
Osteogenic
senchymal Stem Cell O
c Differentia
ation Mediu
um (Cat. No
o. GUXMXOriCellTM Mes
0021)
90
Osteogenes
sis Protoco
ol
Note: The protocol listed below iss for 6-welll tissue cultture platess.
N
M
Rabbit AD
DSCs in OriCellTM Mese
enchymal S
Stem Cell Growth
G
1.. Culture the OriCellTM
a 37°C in a 5% CO2 humidified incubator.
Medium at
3.. Reseed th
he ADSCs in the grow
wth medium
m at 2×104 cells/cm2 iin a 6-well tissue
culture plate pre-co
oated with 0
0.1% gelatin solution.
4.. Incubate the cells at 37°C in a 5% CO2 humidified
h
incubator.
5.. When cells are apprroximately 60-70% co
onfluent, carefully asp irate off the growth
al Stem Ce
dd 2 mL of OriCellTM Mesenchyma
M
ell
medium from each well and ad
nic Differentiation Med
dium.
Osteogen
6.. Feed cells
s every 3 days for 2-3
3 weeks by completely
y replacing the medium with
fresh OriC
CellTM Mese
enchymal S
Stem Cell Osteogenic
O
Differentiat
D
tion Medium
m (prewarmed to
t 37°C).
7.. After 2-3 weeks of differentiati
d
ion, cells ca
an be fixed and staine
ed with alizarin red S.
IMPI0022A2 RB
BXMD‐01001
Page 8 of 15
event osteoblasts from
m detaching
g, it is recommended tto change half
h
of the
No
ote: To pre
me
edium everry two days
s before ana
alysis.
Allizarin Red
d S Stainin
ng Analysiis
ve the osteo
ogenic diffe
erentiation medium
1.. After the cells have differentiatted, remov
from the wells and rinse with 1
1x phospha
ate-buffered
d saline (PB
BS). Fix ce
ells with 2
mL of 4%
% formaldeh
hyde soluti on for 30 minutes.
m
2.. Rinse wells twice with 1x PBS. Stain the cells with 1 mL alizarrin red S wo
orking
f
for
3-5
min
nutes.
solution
3.. Rinse wells 2-3 time
es with 1x P
PBS.
4.. Cells can now be vis
sualized an
nd analyzed
d under a microscope.
m
TM
Fig
g. 3 OriCell Rabbit
R
Adipose
e-Derived Mes
senchymal Ste
em Cells are d
differentiated to
Osteocytess and are stain
ned with Alizarin Red S.
Adipogenic Differen
ntiation
Materials Required
R
M
senchymal Stem Cell A
Adipogenic
c Differentia
ation Mediu
um (Cat. No
o. GUXMXOriCellTM Mes
0031)
90
Ad
dipogenes
sis Protoco
ol
Note: The protocol listed below iss for 6-welll tissue cultture platess.
N
1.. Culture the
t
OriCellTM Rabbit AD
DSCs in the
e OriCellTM Mesenchym
M
mal Stem Cell Growth
Medium at
a 37°C in a 5% CO2 humidified incubator.
2.. When cells are apprroximately 80-90% co
onfluent, th
hey can be dissociated
d with
psin-0.04%EDTA (Cat. N
No. TEDTA-1000).
0.25%Tryp
3.. Reseed the ADSCs in growth m
medium at 2x104 cells
s/cm2 in a 6
6-well tissue culture
m volume o
of 2 mL perr well.
plate with a medium
4.. Incubate
e the cells at
a 37°C in a 5% CO2 humidified
h
incubator.
5.. Feed the cells every
y three day
ys until they
y are 100%
% confluent or post-confluent.
IMPI0022A2 RB
BXMD‐01001
Page 9 of 15
Induction of adipogenic differentiation at post-confluency is strongly recommended.
6. When the cells are 100% confluent or post-confluent, carefully aspirate off the
spent growth medium from the wells and add 2 mL of OriCellTM Mesenchymal Stem
Cell Adipogenic Differentiation medium A (induction medium) per well.
7. Three days later, change the medium to OriCellTM Mesenchymal Stem Cell
Adipogenic Differentiation medium B (maintenance medium) by completely
replacing the spent medium A.
8. 24 hours later, change the medium back to MSC Adipogenic Differentiation medium
A.
9. To optimally differentiate MSCs into adipogenic cells, repeat the cycle of induction
and maintenance three times.
10. After three to five cycles of induction and maintenance, culture the cells in OriCellTM
Mesenchymal Stem Cell Adipogenic Differentiation medium B for an additional 4-7
days until the lipid droplets are big, round enough. During these days period,
change the medium every three days.
Oil Red O Stain Analysis
1. After the cells have differentiated, remove the MSC Adipogenic Differentiation
Medium from the wells and rinse with 1x phosphate-buffered saline (PBS). Fix cells
with 2 mL of 4% formaldehyde solution for 30 minutes.
2. Rinse wells twice with 1x PBS and stain cells with 1 mL of oil red O working solution
(3:2 dilution with distilled water and filter with filter paper) for 30 minutes.
3. Rinse wells 2-3 times with 1x PBS.
4. Cells can now be visualized and analyzed under a microscope.
TM
Fig.4 OriCell Rabbit Adipose-Derived Mesenchymal Stem Cells are differentiated to adipocytes and are
stained with Oil Red O.
Chondrogenic Differentiation
Materials Required
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium
(Cat. No. GUXMX-90041)
IMPI0022A2 RBXMD‐01001
Page 10 of 15
Chondrogenesis Protocol
1. Calculate the total number of ADSC pellet cultures required for your experiment
(2.5×105 ADSCs are needed to form each chondrogenic pellet). Transfer this
amount of cells into an appropriate culture tube.
2. Wash the ADSCs with Incomplete Chondrogenic Medium. Centrifuge the cells at
150 x g for 5 minutes at room temperature, and then aspirate off the supernatant.
Resuspend the cells in 1 mL of Incomplete Chondrogenic Medium per 7.5×105 cells.
Centrifuge again at 150 x g for 5 minutes, and then aspirate off the medium.
3. Resuspend the ADSCs in Complete Chondrogenic medium to a concentration of
5.0×105 cells/mL.
4. Aliquot 0.5 mL (2.5×105 cells) of the cell suspension into 15 mL polypropylene
culture tubes. Centrifuge the cells at 150 x g for 5 minutes at room temperature.
DO NOT aspirate the supernatant nor resuspend the pellet.
5. Loosen the caps of the tubes in order to allow gas exchange and incubate the tubes
at 37°C in a humidified atmosphere of 5% CO2. Do not disturb the pellets for 24
hours.
6. Feed the cell pellets every 2-3 days by completely replacing the medium in each
tube (to avoid aspirating the pellets when aspirating the medium, attach a sterile 1200μL pipette tip to the end of the aspirating pipette). Add 0.5 mL of freshly
prepared Complete Chondrogenic Medium to each tube.
7. After replacing the medium, flick the bottom of the tube to ensure that the pellet is
free floating. Loosen the caps and return the tubes to the 37°C incubator.
8. Chondrogenic pellets should be harvested after 14-28 days in culture. Pellets may
be formalin-fixed and paraffin-embedded for alcian blue stain analysis.
IMPI0022A2 RBXMD‐01001
Page 11 of 15
Allcian Blue Staining Procedure
e
1.
ue sample should
s
be fo
ormalin-fixed and paraffin-embe
edded already.
The tissu
2.
Staining procedure::
a)
Dep
paraffinize slides and hydrate to distilled wa
ater.
b)
Sta
ain in alcian
n blue soluttion for 30 minutes.
c)
Wash in running tap watter for 2 minutes.
d)
Rinse in distilled water.
e)
er a light m
microscope and
a
capture
e images fo
or analysis.. Blue
Visualize unde
ates synthe
esis of prote
eoglycans by
b chondro
ocytes.
staining indica
g.5 OriCellTM Rabbit ADSCs
s are different iated to chond
drocytes and are stained w
with Alcian Blue
e.
Fig
CRYOPR
RESERVA
ATION OF
O CELL
LS USIN
NG OriCe
ellTM
CRYOPR
RESERVA
ATION MEDIA
M
OriCellTM NCR
R Protein-F
Free Cryoprreservation Medium (C
Cat. No. NC
CPF-10001) is a
use freezing
g medium. Its chemically-define
ed and prottein-free
prrotein-free,, ready-to-u
fo
ormulation has
h been optimized to
o stem cells
s and prima
ary cells, th
hus greatly enhancing
th
he viability and integrity of these cells by prrotecting th
hem from da
amage durring the
on
ne-step free
eze-thaw procedure.
p
U
Unlike othe
er conventio
onal freezin
ng media, which
w
re
equire a slow programmed freeze
e, this prod
duct allows the cells to
o be directly
y frozen at 80
0°C.
Cryopreservation
ge the cultu
ure medium
m with fresh
h growth medium
m
24 h
hours before freezing
Note: Chang
g.
N
ells that are
e in the log
garithmic growth phas
se. Perform
m a cell count to
1.. Collect ce
determin
ne the viable cell dens ity.
2.. Centrifug
ge the cells for 3-5 mi nutes at 25
50 x g and 20°C. Rem
move and discard the
supernattant using a pipette.
IMPI0022A2 RB
BXMD‐01001
Page 12 of 15
5
3. Resuspend the cell pellet in the OriCellTM NCR Protein-Free Cryopreservation Medium
at a cell density of 105-106 cells/mL.
4. Dispense aliquots of the cell suspension into cryogenic storage vials that are
properly labeled.
5. Place the vials directly in a -80°C freezer. After 24 hours, transfer the frozen vials
to liquid nitrogen for long-term preservation.
IMPI0022A2 RBXMD‐01001
Page 13 of 15
APPENDIX
Troubleshooting
The table below lists some potential problems and solutions for culturing ADSCs.
Problem
Low cell recovery
rate
Cause
Solution
The storage condition does not
meet the requirements
Purchase a replacement and store in liquid
nitrogen for long‐term preservation.
Thawing of the cells takes too
long
Thaw cells for no more than 3 minutes.
Cells are incompletely
recovered after thawing
After aspirating off medium, wash the tube
with culture medium twice and transfer all
of the cells to the dish.
Cells are handled roughly
Care should be taken to avoid introducing
bubbles during pipetting. Also avoid
vortexing and high‐speed centrifugation
Medium is not pre‐warmed
Warm medium to 37°C before recovery.
Mycoplasma contamination
Discard the cells in question and disinfect
the laboratory environment before
recovering the next batch of cells.
Slow cell growth
Over digestion
Wash the cells with PBS 2‐3 times to remove
serum prior to trypsinization (serum will
inhibit the function of trypsin).
Control the digestion time.
Cell aging
IMPI0022A2 RBXMD‐01001
Plating density is too low
Increase the plating density.
Inappropriate serum and
medium
Use Cyagen tailor‐made culture media. If
other serum and media products are used,
please perform validation to ensure
compatibility.
Dead cells are not removed
promptly
Change the medium next day after recovery
to ensure removal of all dead cells.
Cell Contamination
Discard the cells in question and disinfect
the laboratory environment before
recovering the next batch of cells.
Plating density is too low
Some stem cells can secrete factors to
support cell growth. Therefore, a certain
degree of plating density must be
maintained; otherwise, it will lead to cell
proliferation slow down and cell aging.
Page 14 of 15
R
Cell aging
E
L
A
T
Cells show
E
D spontaneous
differentiation
P Ineffective
R induction of cell
differentiation
Over digestion
Wash the cells with PBS 2‐3 times to remove
serum prior to trypsinization (serum will
inhibit the function of trypsin).
Control the digestion time.
The passaging time is not
appropriate
The cells should be subcultured when
reaching 80‐90% confluency in order to
avoid contact inhibition.
DMSO is not completely
removed during cell recovery
Wash the cells with pre‐warmed medium 2‐
3 times during recovery.
Differentiation reagents need
to be optimized
Cell passage is too high
Use Cyagen tailor‐made differentiation
media.
Use cells at a low original passage number.
RELATED PRODUCTS
Product
Catalog Number
0.25%Trypsin-0.04%EDTA
TEDTA-10001
Phosphate-Buffered Saline (1xPBS)
PBS-10001
OriCellTM Rabbit Adipose-Derived Mesenchymal Stem Cells
RBXMD-01001
OriCellTM Rabbit Adipose-Derived Mesenchymal Stem Cell
Growth Medium
GUXMD-90011
OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation
Medium
GUXMX-90021
OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation
Medium
GUXMX-90031
OriCellTM Mesenchymal Stem Cell Chondrogenic
Differentiation Medium
GUXMX-90041
OriCellTM NCR Protein-Free Cryopreservation Medium
NCPF-10001
REFERENCES
JM Gimble, and F Guilak. (2003) Adipose-derived adult stem cells: isolation,
characterization, and differentiation potential. ISCT 5: 362-369.
Cyagen Biosciences reserves all rights on the technical documents of its OriCellTM cell
culture products. No part of this document may be reproduced or adapted for other
purposes without written permission from Cyagen Biosciences.
IMPI0022A2 RBXMD‐01001
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