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User Manual
OriCellTM Strain C57BL/6 Mouse
Adipose-Derived Mesenchymal
Stem Cells (ADSCs)
Cat. No. MUBMD-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 Strain C57BL/6 Mouse ADSCs
Thawing and Establishing OriCellTM Strain C57BL/6 Mouse ADSCs ……………..…………… 4
Passaging Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs ……………..……………… 6
Differentiation of OriCellTM Strain C57BL/6 Mouse ADSCs …………….………………… 8
Cryopreservation of OriCellTM Strain C57BL/6 Mouse ADSCs ………..………………… 12
Appendix ………………………………………………………………………………… 13
Troubleshooting ……………………………………………………………………………………………… 13
Related Products …………………………………………………………………………………………… 14
References …………………………………………………………………………………………………… 14
CONTENTS AND STORAGE
Product Name
Strain C57BL/6 Mouse Adipose‐
Derived Mesenchymal Stem Cell
Catalog No.
MUBMD‐01001
Amount per Vial
1×106 Cells
Cryopreserved At
Sixth 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. In addition, ADSCs have wide applications in tissue engineering, cell
therapy and gene therapy.
Cyagen OriCellTM Strain C57BL/6 Mouse ADSCs are derived from adipose tissue at inguen
of qualified strain C57BL/6 mice. These cells express clusters of proteins specific for
ADSCs and have a strong capacity for self-renewal 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 3 times.
IMPI0020A2 MUBMD‐01001 Page 3 of 14 •
Multipotent diffferentiation
n ability along osteogenic, chondrrogenic and
d
ogenic linea
ages.
adipo
•
Posittive for CD2
29, CD44, a
and Sca-1 (> 70%), and
a
negativ
ve for CD11
17 (<5%)
in flo
ow cytomettry assays.
PRODUC
CT APPL
LICATIO
ONS
Adipose-Deriv
ved Mesenchymal Stem
m Cells (AD
DSCs) have become a hot research target
ntial use in regenerativ
ve medicine and tissu
ue engineerring (in area
as such as
for their poten
cardiovascularr, neural an
nd orthopae
edic disease
e).
agen OriCelllTM Strain C57BL/6
C
Mo
ouse ADSC
Cs can be us
sed as cell models to evaluate
Cya
the
e immunore
eactions, prroliferation,, immigratio
on and diffe
erentiation of ADSCs both in vivo
o
d in vitro.
and
GENERA
AL HAND
DLING PRINCIP
P
PLES
1.
Aseptic ha
andling of th
he product is necessary througho
out.
2.
Once the cells
c
have been
b
establlished, alwa
ays freeze up several vials of AdiposeDerived Mesenchyma
al Stem Cellls (ADSCs)) as a backu
up.
Notte: The OriiCellTM Strain C57BL/6
6 Mouse AD
DSCs can be
e frozen/th
hawed at least one
tim
mes.
3.
For genera
al maintena
ance of cell s, we recom
mmend the
e seeding d ensity to be 1.02.0×104ce
ells/cm2.
4.
For all stud
dies, it is sttrongly reccommended
d to use cells that are at, or unde
er, an
original pa
assage num
mber of 10.
5.
For genera
al maintena
ance of cell s, we recom
mmend tha
at the medi um is chan
nged if it
becomes acidic
a
(the pH indicato
or in culture
e medium appears
a
yelllow). In ge
eneral,
change the
e growth medium
m
eve
ery three da
ays.
6.
Do not let Strain C57
7BL/6 Mousse ADSCs overgrow, as it will ressult in conta
act
inhibition. When the
e cells are 8
80-90% con
nfluent, sub
bculturing tthe cells is strongly
recommen
nded.
Notte: We stro
ongly recom
mmend the use of OriC
CellTM cultu
ure media a
and other re
elated
rea
agents for optimal
o
resu
ults.
THAWIN
NG AND
D ESTABLISHIN G OriCe
ellTM STRA
AIN C57
7BL/6 MOUSE
M
ADSCs
Materials Required:
•
S
C57B
BL/6 Mouse Adipose-D
Derived Mes
senchymal S
Stem Cells (Cat. No.
OriCellTM Strain
MUBMD-01
1001)
IMPI0020A2 M
MUBMD‐01001 Page 4 of 14
•
OriCellTM Mouse
M
Adipo
ose-Derived
d Mesenchy
ymal Stem Cell Growtth Medium (Cat. No.
MUXMD-90
0011)
Tha
awing and
d Establish
hing Mouse
e ADSCs
1.
Pre-warm OriCellTM Mouse
M
ADSC
C Growth Medium
M
to 37°C.
3
2.
M
Add 9 mL of OriCellTM
Mouse AD
DSC Growth
h Medium to a 15 mL conical tub
be.
3.
M
he cryovial of OriCellTM
Strain C5
57BL/6 Mouse ADSCs ffrom liquid nitrogen.
Remove th
4.
Quickly thaw the cryo
ovial in a 3
37°C water bath until the
t
last ice crystal dis
sappears.
o finish the thawing prrocedure wiithin 3 minutes. Be ca
areful not to
o submerge
e
Be sure to
the entire vial. Maxim
mum cell vi ability is de
ependent on the rapid
d and comp
plete
thawing off frozen cells.
s will be les
ss than optiimal if the cells are th
hawed for m
more than 3 minutes.
Notte: Results
5.
As soon as
s the cells are
a comple tely thawed
d, disinfect the outsid e of the via
al with 70%
%
v/v ethano
ol.
6.
Use a pipe
ette to trans
sfer the cellls to the co
onical tube containing
g OriCellTM Mouse
M
ADSC Grow
wth Medium
m inside a b
biosafety cabinet. Be careful nott to introduce any
bubbles du
uring the trransfer proccess.
7.
Rinse the vial with 1 mL of the medium to reduce cell loss. Sub
bsequently transfer
pension into
o the conica
al tube.
this 1 mL of cell susp
8.
x the cell su
uspension b
by slowly pipetting
p
up
p and down . Be carefu
ul not to
Gently mix
introduce any bubble
es.
9.
e the cell su
uspension a
at 250 x g for
f 5 minutes.
Centrifuge
a
offf as much o
of the supernatant as possible an
nd add 2-3 mL of fresh
h
10. Carefully aspirate
OriCellTM Mouse
M
ADSC
C Growth M
Medium (pre-warmed to 37°C).
he cells in O
OriCellTM Mo
ouse ADSC Growth Me
edium.
11. Gently re-suspend th
c
into a T25 flask a
and add a sufficient
s
OriCellTM Mou
use ADSC Growth
G
12. Plate the cells
Medium. Gently
G
rock the culture
e flask to evenly distribute the ce
ells.
a 37°C in a 5% CO2 h
humidified incubator.
13. Incubate at
d
change
e the mediu
um with fre
esh growth medium (p
pre-warmed
d to 37°C)..
14. The next day,
he growth medium
m
eve
ery three days thereaffter.
15. Change th
pproximate
ely 80-90%
% confluent, they can b
be dissociatted with
16. When the cells are ap
0.25%Trypsin-0.04%
%EDTA and passaged.
um
Notte: Changiing Mediu
1.
Warm an appropriate
a
e amount o f medium to
t 37°C in a sterile con
ntainer. Re
eplace the
spent med
dium with the pre-warrmed, fresh
h medium. Once comp
pleted, retu
urn the
flask to the incubator.
2.
eated warm
ming and co
ooling of the medium. If the entiire contentt is not
Avoid repe
needed forr a single procedure,
p
ttransfer on
nly the requ
uired volum
me to a ster
rile
secondary
y container..
IMPI0020A2 M
MUBMD‐01001 Page 5 of 14
TM
Fig. 1 OriCell
Strain C57BL/6 Mouse Adipose-Derived Mesenchymal Stem Cells are established.
PASSAGING OriCellTM STRAIN C57BL/6 MOUSE ADSCs
Materials Required:
•
0.25%Trypsin-0.04%EDTA (Cat. No. TEDTA-10001)
• Phosphate-Buffered Saline (1× PBS) (Cat. No. PBS-10001)
• OriCellTM Strain C57BL/6 Mouse Adipose-Derived Mesenchymal Stem Cells
(Cat. No. MUBMD-01001)
• OriCellTM Mouse Adipose-Derived Mesenchymal Stem Cell Growth Medium
(Cat. No. MUXMD-90011)
Passaging Mouse ADSC
1.
Pre-warm the OriCellTM Mouse ADSC Growth Medium, 1×PBS, and 0.25%Trypsin0.04%EDTA solution to 37°C.
2.
Carefully aspirate spent medium from the 80-90% confluent monolayer of OriCellTM
Strain C57BL/6 Mouse ADSCs.
3.
Add 1×PBS (6 mL for T25 flask, 3 mL for T25 flask). Be careful not to disturb the
monolayer. Gently rock the flask back and forth to rinse the monolayer.
4.
Aspirate 1× PBS and discard.
5.
Repeat the step 3-4 two or three times.
6.
Add 0.25%Trypsin-0.04%EDTA solution (2-3 ml for T75 flask, 1 ml for T25 flask).
Gently rock the flask back and forth to ensure that the entire monolayer is covered
with the 0.25%Trypsin-0.04%EDTA solution. Allow trypsinization to continue until
the majority of the cells (approximately 80%) are rounded up. At this point, gently
tap the side of the flask to release the majority of cells from the culture surface.
Important: Avoid leaving cells exposed to the trypsin longer than necessary (no
more than two minutes if using Cyagen’s trypsin-EDTA solution). Care should also
be taken that the cells are not forced to detach prematurely as this may result in
clumping.
7.
After the cells are visibly detached, immediately add the pre-warmed OriCellTM Mouse
IMPI0020A2 MUBMD‐01001 Page 6 of 14 ADSC Grow
wth Medium
m (6 ml forr T75 flask, 3 ml for T25 flask) to
o neutralize
e the
trypsinizattion.
8.
Gently pip
pet the med
dium over tthe cells to dislodge an
nd resuspe nd the cells
s. Repeat
5-6 times until all the
e cells are d
dissociated
d from the flask
f
and ev
venly dispe
ersed into a
single cell suspension
n.
9.
Transfer th
he dissociated cells in to a 15 mL
L conical tub
be.
10. Centrifuge
e at 250 x g for 5 minu
utes to pelllet the cells
s.
a
offf as much o
of the supernatant as possible.
11. Carefully aspirate
o OriCellTM Mouse AD SC Growth Medium to
o the conica
al tube and
d gently re-12. Add 2 ml of
suspend th
he cells tho
oroughly.
c
into ap
ppropriate fflasks. OriC
CellTM Strain
n C57BL/6 Mouse ADS
SCs can be
13. Plate the cells
split at 1:2
2 or other appropriate
a
e ratio.
a
of m
medium to the cells. Incubate th e cells at 37°C
3
inside
14. Add an appropriate amount
a 5% CO2 humidified incubator..
hould be tak
ken to avoiid introduciing bubbles
s during pip
petting.
Notte: Care sh
Add
ditional Tips
T
Tim
me to Chan
nge Mediu
um
It is
s recomme
ended to ch
hange the cculture med
dium if therre are too m
many dead cells after
pas
ssaging.
It is
s recomme
ended to ch
hange the cculture med
dium whene
ever the m
medium becomes acidic,
eve
en if the cells do not reach
r
80-90
0% conflue
ency. The pH
p indicato
or in the culture
medium will appear
a
yello
ow when accidic. In ge
eneral, change the gro
owth mediu
um every
ee days.
thre
me to Subc
culture
Tim
T
Strain C5
When OriCellTM
57BL/6 Mou
use ADSCs
s are 80-90
0% conflue nt, it is rec
commended
d
ured. Do no
ot let OriCe
ellTM Strain C57BL/6 M
Mouse ADSC
Cs overgrow
w
that the cells be subcultu
as iit will result in contactt inhibition..
P
Passage
9-40x
TM
Fig.. 2 Images off OriCell
Passa
age 9-100x
Stra
ain C57BL/6 M
Mouse Adipose
e-Derived Mesenchymal Ste
em Cells at pa
assage 9.
IMPI0020A2 M
MUBMD‐01001 Page 7 of 14
OriCellTTM STRAI
IN C57B
BL/6 MO
OUSE AD
DSCs DIFFEREN
NTIATION
TM
USING O
OriCell DIFFERENTIA
ATION MEDIA
M
Cya
agen OriCellTM ADSCs can differe
entiate into a variety of
o cell typess, including
g osteocytes,
adip
cytes.
pocytes and chondroc
Osteogenic Differenttiation
Materials Required:
CellTM Mese
enchymal Stem
S
Cell O
Osteogenic Differentiat
D
ion Medium
m (Cat. No. GUXMXOriC
900
021)
Ostteogenesis Protocoll
otocol listed
d below is ffor 6-well tissue
t
cultu
ure plates.
Notte: The pro
1.
Culture the OriCellTM Starin C57
7BL/6 Mous
se ADSCs in
n OriCellTM M
Mouse ADS
SC Growth
humidified incubator.
Medium att 37°C in a 5% CO2 h
2.
When cells
s are appro
oximately 8
80-90% con
nfluent, they can be diissociated with
w
0.25%Trypsin-0.04%
%EDTA (Catt. No. TEDT
TA-10001).
3.
e OriCellTM Starin C57
7BL/6 Mouse ADSCs in
n the growtth medium at
Reseed the
4
3×10 cells
s/cm2 in a 6-well
6
tissu
ue culture with
w
a medium volume
e of 2 mL per
p well.
4.
Incubate the
t
cells at 37°C in a 5
5% CO2 hu
umidified incubator.
5.
When cells
s are appro
oximately 6
60-70% con
nfluent, carefully aspirrate off the growth
medium frrom each well
w and add
d 2 mL of OriCellTM
O
Mesenchyma
M
al Stem Cell
Osteogenic Differentiiation Medi um (pre-wa
armed to 37°C).
6.
ays for 2-3 weeks by completely
c
replace the
e medium with
w
fresh
Feed cells every 3 da
OriCellTM Mesenchym
M
ell Osteogen
nic Differen
ntiation Med
dium.
al Stem Ce
7.
After 2-3 week
w
differentiation, ccells can be
e fixed and stained witth Alizarin Red S.
vent osteoblasts from detaching, it is recom
mmended to
o change ha
alf of the
Notte: To prev
med
dium every
y two days before
b
anallysis.
zarin Red Stain Ana
alysis
Aliz
1.
After the cells
c
have differentiate
d
ed, remove
e the osteog
genic differrentiation medium
m
from the wells
w
and rinse with 1x
x phosphatte-buffered saline (PBS
S). Fix cellls with 2
mL of 4% formaldehy
yde solutio n for 30 minutes.
2. Rinse wells
s twice with
h 1x PBS. S
Stain the ce
ells with 1 mL alizarin red S working
s
solution forr 3-5 minuttes.
3. Rinse wells
s 2-3 times with 1x PB
BS.
Cells can now be visualized and analyzed under
u
a mic
croscope.
4. C
IMPI0020A2 M
MUBMD‐01001 Page 8 of 14
TM
Fig
g. 3 OriCell
Strain C57BL//6 Mouse Adip
pose-derived Mesenchymal Stem Cells a re differentiatted into
Osteocytes
O
and
d are stained with Alizarin Red
R S.
Adipogenic Differenttiation
Materials Required
CellTM Mese
enchymal Stem
S
Cell A
Adipogenic Differentia
ation Mediu
um (Cat. No.
N GUXMX
XOriC
900
031)
Adiipogenesis
s Protocoll
otocol listed
d below is ffor 6-well tissue
t
cultu
ure plates.
Notte: The pro
1.
Culture the OriCellTM Strain C57
7BL/6 Mous
se ADSCs in
n the OriCe llTM Mouse ADSC
edium at 37
7°C in a 5%
% CO2 humidified incubator.
Growth Me
2.
When cells
s are appro
oximately 8
80-90% con
nfluent, they can be diissociated with
w
0.25%Trypsin-0.04%
%EDTA (Catt. No. TEDT
TA-1000).
3.
e ADSCs in
n growth me
edium at 2x104 cells/c
cm2 in a 6--well tissue culture
Reseed the
plate with a medium
m volume o
of 2 mL perr well.
4.
t
cells at 37°C in a 5
5% CO2 hu
umidified incubator.
Incubate the
5.
Feed the cells
c
every three
t
days until they are 100% confluent o
or post-confluent.
Induction of adipogen
nic differen
ntiation at post-conflue
p
ency is stro
ongly recom
mmended.
6.
00% conflu
uent or post-confluentt, carefully aspirate offf the spentt
When the cells are 10
growth me
edium from
m the wells a
nchymal Ste
em Cell
and add 2 mL of OriCellTM Mesen
Adipogenic
c Differentiation medi um A (induction med
dium) per w
well.
7.
TM
Three days later, cha
ange the m edium to OriCell
O
Me
esenchymall Stem Cell Adipogenic
c
ation mediu
um B (main
ntenance medium) by completely
y replacing the spent
Differentia
medium A.
A
8.
24 hours later, chang
ge the med
dium back to
t MSC Adip
pogenic Diffferentiation
n medium A.
A
9.
ogenic cells
s, repeat th
he cycle of induction
To optimally differenttiate ADSC s into adipo
and maintenance thrree times.
e to five cycles of indu
uction and maintenanc
ce, culture the cells in
n OriCellTM
10. After three
Mesenchym
mal Stem Cell
C Adipoge
entiation medium B fo
or an additio
onal 4-7
enic Differe
days until the lipid drroplets are big, round enough. During
D
these
e days period, change
e
IMPI0020A2 M
MUBMD‐01001 Page 9 of 14
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
Strain C57BL/6 Mouse Adipose-derived Mesenchymal Stem Cells are differentiated into
adipocytes and are stained with Oil Red O.
Chondrogenic Differentiation
Materials Required:
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium
(Cat. No. GUXMX90041)
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
IMPI0020A2 MUBMD‐01001 Page 10 of 14 culture tub
bes. Centrifuge the ce
ells at 150 x g for 5 minutes
m
at rroom temperature.
T aspirate the
t
superna
atant or res
suspend the pellet.
Notte: DO NOT
5.
d incubate the tubes
e caps of th
he tubes in order to allow gas ex
xchange and
Loosen the
at 37°C in a humidified atmosph
% CO2. Do not
n disturb the pellets
s for 24
here of 5%
hours.
6.
c pellets every
e
2-3 d
days by com
mpletely re
eplacing the
e medium in each tube
e
Feed the cell
(to avoid aspirating
a
the
t
pellets w
when aspirrating the medium,
m
atttach a sterile 1-200μL
L
pipette tip
p to the end
d of the asp
pirating pipette). Add 0.5 mL of freshly pre
epared
Complete Chondroge
enic Medium
m to each tu
ube.
7.
acing the medium,
m
flicck the botto
om of the tu
ube to ensu
ure that the
e pellet is
After repla
free floatin
ng. Loosen
n the caps a
and return the tubes to
t the 37°C
C incubator.
8.
Chondroge
enic pellets should be harvested after 14-28
8 days in c ulture. Pellets may be
e
formalin-fiixed and pa
araffin-emb
bedded for alcian blue stain analy
ysis.
Alc
cian Blue Staining
S
Procedure
1.
The tissue
e sample sh
hould be forrmalin-fixed
d and parafffin-embed ded already.
2.
Staining procedure:
affinize slide
es and hyd rate to disttilled water.
a) Depara
b) Stain in
n alcian blue solution ffor 30 minu
utes.
c) Wash in
n running tap water fo
or 2 minute
es.
d) Rinse in
n distilled water.
w
nalysis. Blue staining
e) Visualiz
ze under a light micro
oscope and capture im
mages for an
g
indicates synthesis of proteo
oglycans by
y chondrocy
ytes.
TM
F
Fig.5 OriCell
Strain C57BL
L/6 Mouse Adi pose-Derived Mesenchymal Stem Cells a
are differentia
ated
into chondrocytes and are stain
ned with Alcian
n Blue.
IMPI0020A2 M
MUBMD‐01001 Page 11 of 14
4 CRYOPR
RESERVA
ATION OF
O OriC ellTM STRAIN C5
57BL/6 MOUSE
E
ADSCs
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
Unlike other conventiional freezi ng media, which
re
equire a slow programmed freeze
e, this prod
duct allows the cells to
o be directly
y frozen at
-8
80°C.
Cryopreservation
m with fres
sh growth medium
m
24 hours befo
N
Note: Change the cultture medium
ore freezing
g.
1
1.
Collect cells that are
a in the lo
ogarithmic growth pha
ase. Perforrm a cell co
ount to
ble cell den
nsity.
determine the viab
2
2.
Centrifu
uge the cells for 3-5 m
minutes at 250
2
x g and
d 20°C. Re move and discard the
e
superna
atant using a pipette.
3
3.
Resuspend the celll pellet in tthe OriCellT
TM NCR Pro
otein-Free C
Cryopreserv
vation
m at a cell density
d
of 1
105-106 cells/mL.
Medium
4
4.
Dispens
se aliquots of the cell suspension
n into cryog
genic storag
ge vials tha
at are
properly
y labeled.
5
5.
Place th
he vials dire
ectly in a -8
80°C freeze
er. After 24 hours, tra
ansfer the frozen vials
s
to liquid
d nitrogen for
f long-terrm preservation.
IMPI0020A2 M
MUBMD‐01001 Page 12 of 14
4 APPE
NDI
X
Cell aging
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 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
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 the 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. IMPI0020A2 MUBMD‐01001 Page 13 of 14 proliferation slow down and cell aging.
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. Cells show
spontaneous
differentiation
Ineffective
induction of cell
differentiation
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
OriCellTM Mouse Adipose-Derived Mesenchymal
Stem Cell Growth Medium
MUXMD-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
0.25%Trypsin-0.04%EDTA
TEDTA-10001
Phosphate-Buffered Saline (1xPBS)
PBS-10001
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.
IMPI0020A2 MUBMD‐01001 Page 14 of 14