Download User Manual - Cyagen Biosciences

 User Manual
OriCellTM Fischer 344(F344) Rat
Mesenchymal Stem Cells with RFP
(MSCs/RFP)
Cat. No. RAFMX-01201
Table of Contents
Contents and Storage ……………………………………………………………………………………… 3
Product Introduction ……………………………………………………………………………………… 3
Cell Characteristics and Identity ……………………………………………………………………… 3
Product Application …………………………………………………………………………………………4
General Handling Principles ……………………………………………………………………………4
Culturing OriCellTM Fischer 344(F344) Rat MSCs/RFP
Thawing and Establishing OriCellTM Fischer 344(F344) Rat MSCs/RFP ……………………4
Passaging Cyagen OriCellTM Fischer 344(F344) Rat MSCs/RFP ………………………………6
Differentiation of OriCellTM Fischer 344(F344) Rat MSCs/RFP …………………………………7
Cryopreservation of OriCellTM Fischer 344(F344) Rat MSCs/RFP ……………………………11
Appendix ………………………………………………………………………………… 13
Troubleshooting ……………………………………………………………………………………………… 13
Related Products …………………………………………………………………………………………… 14
References …………………………………………………………………………………………………… 14
Technical Support ……………………………………………………………….…… 15
CONTENTS AND STORAGE
Product Name
Fischer 344(F344) Rat
Mesenchymal Stem Cells
Catalog No.
RAFMX‐01201
Amount per Vial
1×106 Cells
Cryopreserved At
Fifth 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
Mesenchymal stem cells (MSCs/RFP) are multipotent stem cells that can differentiate
into a variety of cell types including osteocytes, adipocytes, and chondrocytes.
MSCs/RFP proliferate quickly and are capable of generating a local immunosuppressive
microenvironment, thus contributing to their wide application potentials in tissue
engineering, cell therapy, and gene therapy.
OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells are derived from the bone
marrow of Fischer 344(F344) Rats, cultured as monolayer, and then have been
transfected with a lentiviral construct containing a RFP expression motif.
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
IMPI0075A2 RAFMX‐01201 Page 3 of 15 adipo
ogenic linea
ages.
•
Posittive for CD2
29, CD44, a
and CD90 (>
( 70%), and
a
negativ
ve for CD34
4 and
CD45
5 and CD11
1b (< 5%) in flow cytometry ass
says.
PRODUC
CT APPL
LICATIO
ONS
Fischer 344 (F344) Rat MSCs/RFP
P have beco
ome a popular researcch target du
ue to their
otential use
e in regenerative med icine and tiissue engin
neering (in areas such as
po
ca
ardiovascula
ar, neural, and orthop
pedic diseas
se).
cher 344 (F
F344) Rat M
MSCs/RFP can
c
be used
d as cell mo
odels to eva
aluate the
OriCellTM Fisc
mmunoreactions, proliferation, im
mmigration,, and differentiation o f MSCs/RFP
P both in
im
viivo and in vitro.
v
GENERA
AL HAND
DLING PRINCIP
P
PLES
h
of the producct is necess
sary througho
out.
1.. Aseptic handling
2.. Once the
e cells have been estab
blished, alw
ways freeze
e several vi als of OriCe
ellTM
Fischer 344
3
(F344) Rat MSCs/ RFP as a ba
ackup.
Note: The O
OriCellTM Fischer 344(F
F344) Rat MSCs/RFP
M
can
c
be froze
en/thawed
d at least
N
tw
wo times.
ecommende
ed to use ce
ells that are
e at, or und
der, an
3.. For all studies, it is strongly re
p
number of 10
0.
original passage
4.. For general mainten
nance of ce
ells, we reco
ommend th
he seeding density to be 2.02
c
.
3.0×104cells/cm
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 the medium
m
app
pears yellow
w). In general,
becomes acidic (the
very three days.
d
change the growth medium ev
6.. Do not le
et OriCellTM Fischer 344
4(F344) Ra
at MSCs/RF
FP overgrow
w as it will result
r
in
contact in
nhibition. When
W
the ce
ells are 80--90% confluent, subcu
ulturing the
e cells is
strongly recommend
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
NG AND
D ESTABLISHIN G OriCe
ellTM FIS
SCHER 3
344(F34
44) RAT
T
MSCs/R
RFP
M
Materials Required
R
•
OriCellTM Mesenchym
mal Stem C
Cell Growth Medium (C
Cat. No. GU
UXMX-9001
11)
IMPI0075A2 RA
AFMX‐01201 Page 4 of 15 Th
hawing and
a
Estab
blishing F ischer 34
44(F344) Rat MSC s/RFP
m the fully supplemen
s
ted (complete) OriCelllTM MSC Grrowth Medium to
1.. Pre-warm
37°C.
2.. Add 9 mL
L of OriCellTM MSC Gro
owth Mediu
um to a 15 mL conical tube.
3.. Remove the cryovia
al of OriCelllTM Fischer 344(F344) Rat MSCs//RFP from liquid
nitrogen..
4.. Quickly thaw
t
the cryovial in a 37°C water bath until the last icce crystal disappears.
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
M
cell viability
y is depende
ent 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
a the cells
s are complletely thawed, disinfec
ct the outsiide of the cryovial
c
5.. As soon as
with 70%
% v/v ethan
nol.
TM
6.. Use a pip
pette to transfer the ccells to the 15 mL coniical tube co
ontaining OriCell
O
MSC Growth Medium
m inside a biosafety cabinet.
c
Be careful nott to introduce any
d
the transfer prrocess.
bubbles during
7.. Rinse the
e vial with 1 mL of the
e medium to
t reduce cell loss. Su bsequently
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 carefful not to
introduce
e any bubbles.
9.. Centrifug
ge the cell suspension
s
at 250 x g for 5 minu
utes.
10
0. Carefully
y aspirate off as much of the supernatant as
s possible a
and add 2-3
3 mL of
fresh OriCellTM MSC Growth Me
edium (pre-warmed to
o 37°C).
11
1. Gently re
esuspend th
he cells in O
OriCellTM MS
SC Growth Medium.
12
2. Seed the
e cells into a T25 flask
k and add a sufficient amount
a
of OriCellTM MSC
M
Growth
h
Medium. Gently roc
ck the cultu
ure flask to
o evenly dis
stribute the
e cells.
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 three days thereafter.
16
6. When the
e cells are approximattely 80-90%
% confluent, they can
n be dissociated with
Trypsin-E
EDTA and passaged.
p
Note: Chang
ging Mediium
N
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.
IMPI0075A2 RA
AFMX‐01201 Page 5 of 15 Fig. 1. OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells with RFP are established.
PASSAGING OriCellTM FISCHER 344(F344) RAT MSCs/RFP
Materials Required
•
0.25%Trypsin-0.04%EDTA (Cat. No. TEDTA-10001)
•
Phosphate-Buffered Saline (1×PBS) (Cat. No. PBS-10001)
•
OriCellTM Fischer 344(F344) Rat Mesenchymal Stem Cells with RFP (Cat. No. RAFMX01201)
•
OriCellTM Mesenchymal Stem Cell Growth Medium (Cat. No. GUXMX-90011)
Passaging OriCellTM Fischer 344(F344) Rat MSCs/RFP
1. Pre-warm the OriCellTM MSC Growth Medium, 1×PBS, and Trypsin-EDTA solution to
37°C.
2. Carefully aspirate the spent medium from the 80-90% confluent monolayer of
MSCs/RFP.
3. Add 1×PBS (6 mL for T75 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 off and discard.
5. Repeat steps 3-4 two or three times.
6. Add 0.25%Trypsin-0.04% 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 Trypsin-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 flask 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 MSC
IMPI0075A2 RAFMX‐01201 Page 6 of 15 Growth Medium
M
(6 mL for T75
5 flask, 3 mL for T25 flask) to neu
utralize the
e
trypsiniza
ation.
8.. Gently piipette the medium
m
ove
er the cells to dislodge
e and resusspend the cells.
c
Repeat 5-6 times un
ntil all the ccells are dis
ssociated frrom the fla
ask and eve
enly
d into a single cell susspension.
dispersed
9.. Transfer the dissocia
ated cells in
nto a 15 mL
m conical tu
ube.
10
0. Centrifug
ge at 250 x g for 5 min
nutes.
11
1. Carefully aspirate off as much of the supe
ernatant as
s possible.
T
MSC Gro
12
2. Add 2 mL
L of OriCellTM
owth Mediu
um to the co
onical tube
e and gently
y resuspend
d
the cells thoroughly
y.
13
3. Plate the cells into appropriate
a
e flasks. OriCellTM Fisc
cher 344(F3
344) Rat MSCs/RFP
plit at 1:2 or
o other ap propriate ratios.
can be sp
14
4. Add an appropriate amount off medium to
o the cells. Incubate tthe cells att 37°C
5 CO2 hu
umidified in cubator.
inside a 5%
N
Note: Care should be taken
t
to av
void introdu
ucing bubbles during pipetting.
Additional Tips
Tiime to Cha
ange Mediium
It is recomm
mended to change
c
the culture me
edium if the
ere are too many dead
d cells afterr
pa
assaging.
It is recomm
mended to change
c
the culture me
edium when
never the m
medium bec
comes
ac
cidic, even if the cells do not rea ch 80-90%
% confluency
y. The pH iindicator in the culture
e
m
medium will appear yellow when a
acidic.
bculture
Tiime to Sub
W
When OriCelllTM Fischer 344(F344)) Rat MSCs//RFP are 80-90% con
nfluent, it is
i
re
ecommende
ed that the
e cells be su
ubcultured.. Do not lett the cells o
overgrow as
s it will
re
esult in contact inhibition.
OriCellTTM FISCH
HER 344
4(F344) RAT MS
SC DIFFE
ERENTIA
ATION USING
U
T
TM
OriCell DIFFE
ERENTIA
ATION M
MEDIA
cher 344(F3
344) Rat M
MSCs/RFP ca
an differenttiate into a variety of cell types
OriCellTM Fisc
dipocytes, and chondrrocytes.
including ostteocytes, ad
O
Osteogenic Differen
ntiation
M
Materials Required
R
senchymal Stem Cell O
Osteogenic
c Differentia
ation Mediu
um (Cat. No
o. GUXMXOriCellTM Mes
0021)
90
IMPI0075A2 RA
AFMX‐01201 Page 7 of 15 sis Protoco
ol
Osteogenes
Note: The protocol listed below iss for 6-welll tissue cultture platess.
N
t
OriCellTM Fischer 3 44(F344) Rat
R MSCs/R
RFP in OriCe
ellTM Mesen
nchymal
1.. Culture the
Stem Cell Growth Medium
M
at 3
37°C in a 5%
5 CO2 hum
midified inccubator.
2.. When cellls are apprroximately 80-90% co
onfluent, they can be d
d with
dissociated
0.25%Try
ypsin-0.04%EDTA (Ca
at. No. TED
DTA-10001)
).
2
3.. Reseed the MSCs in
n the growt h medium at 3×104 cells/cm
c
in a 6-well tissue
oated with 0
0.1% gelatin solution.
culture plate pre-co
4.. Incubate the cells at 37°C insi de a 5% CO2 humidifiied incubattor.
5.. When cellls are apprroximately 60-70% co
onfluent, ca
arefully asp
pirate off the growth
medium from each well and ad
dd 2 mL of OriCellTM Mesenchyma
M
ell
al Stem Ce
nic Differentiation Med
dium.
Osteogen
6.. Feed cells every thrree days forr 2-4 weeks by completely replaccing the medium with
h
CellTM Mese
enchymal S
Stem Cell Osteogenic
O
Differentiat
D
tion Medium
m (prefresh OriC
warmed to
t 37°C).
7.. After 2-4
4 weeks of differentiat
d
ion, cells ca
an be fixed and staine
ed with aliz
zarin red S.
event osteoblasts from
m detaching
g, it is recommended tto change half
h
of the
ote: To pre
No
me
edium everry two days
s before ana
alysis.
d S Stainin
ng Analysiis
Allizarin Red
ve the osteo
ogenic diffe
erentiation medium
1.. After the cells have differentia ted, 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 we
ells twice wiith 1x PBS. Stain the cells with 1 mL alizarrin red S wo
orking
solution for
f 3-5 min
nutes.
3.. Rinse we
ells 2-3 time
es with 1x PBS.
4.. Cells can now be vis
sualized an
nd analyzed
d under a microscope.
m
IMPI0075A2 RA
AFMX‐01201 Page 8 of 15 Fig
g. 3 OriCellTM Fischer 344(F
F344) Rat MSCs/RFP are differentiated in
nto osteocytes
and a
are stained wiith alizarin red
d S.
Adipogenic Differen
ntiation
M
Materials Required
R
OriCellTM Mes
senchymal Stem Cell A
Adipogenic
c Differentia
ation Mediu
um (Cat. No
o. GUXMX90
0031)
Ad
dipogenes
sis Protoco
ol
Note: The protocol listed below iss for 6-welll tissue cultture platess.
N
1.
Culture the
t
OriCellTM Fischer 3 44(F344) Rat
R MSCs/R
RFP in the O
OriCellTM Me
esenchymal
Stem Cell Growth Medium
M
at 3
37°C in a 5%
5 CO2 hum
midified inccubator.
onfluent, they can be d
d with
dissociated
2.. When cellls are apprroximately 80-90% co
0.25%Try
ypsin-0.04%EDTA (Ca
at. No. TED
DTA-1000).
3.. Reseed the MSCs in
n growth me
edium at 2x104 cells/c
cm2 in a 6--well tissue culture
h a medium
m volume o
of 2 mL per well.
plate with
4.. Incubate the cells at 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.
Induction
n of adipoge
enic differe
entiation at post-confluency is strrongly reco
ommended..
6.. When the
e cells are 100% conffluent or po
ost-confluen
nt, carefully
y aspirate off
o the
spent gro
owth mediu
um from the
e wells and
d add 2 mL of OriCellTMM Mesenchy
ymal Stem
Cell Adipogenic Differentiation medium A (induction medium) per well.
7.. Three days later, ch
hange the m
medium to OriCellTM Mesenchyma
M
al Stem Cell
nic Differentiation med
dium B (ma
aintenance medium) b
by complete
ely
Adipogen
replacing
g the spent medium A .
8.. 24 hours later, chan
nge the me
edium back to MSC Ad
dipogenic D
Differentiatio
on medium
m
A.
9.. To optimally differentiate MSC
Cs into adipo
ogenic cells
s, repeat th
he cycle of induction
ntenance att least three
e times.
and main
M
10
0. After thre
ee to five cycles of ind
duction and
d maintenance, culture
e the cells in OriCellTM
Mesenchy
ymal Stem Cell Adipog
genic Differentiation medium
m
B ffor an addittional 4-7
days until the lipid droplets
d
are
e big, round enough. During the se days period,
m every thre
ee days.
change the medium
Oil Red O Stain
S
Analy
ysis
e cells have differentia
ated, remov
ve the MSC maintenan
nce medium
m from the
1.. After the
wells and
d rinse with
h 1x phosph
hate-bufferred saline (PBS). Fix ccells with 2 mL of 4%
formalde
ehyde solution for 30 m
minutes.
2.. Rinse we
ells twice with 1x PBS and stain cells
c
with 1 mL of oil rred O workiing solution
n
IMPI0075A2 RA
AFMX‐01201 Page 9 of 15 (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
Fischer 344(F344) Rat MSCs/RFP are differentiated into adipocytes
and are stained with oil red O.
Chondrogenic Differentiation
Materials Required
OriCellTM Mesenchymal Stem Cell Chondrogenic Differentiation Medium
(Cat. No. GUXMX-90041)
Chondrogenesis Protocol
1. Calculate the total number of MSC pellet cultures required for your experiment
(2.5×105 MSCs/RFP are needed to form each chondrogenic pellet). Transfer this
amount of cells into an appropriate culture tube.
2. Wash the MSCs/RFP 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 MSCs/RFP 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 or resuspend the pellet.
5. Loosen the caps of the tubes one half turn 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.
IMPI0075A2 RAFMX‐01201 Page 10 of 15 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.
Alcian Blue Staining Procedure
1. The tissue sample should be formalin-fixed and paraffin-embedded already.
2. Staining procedure:
a) Deparaffinize slides and hydrate to distilled water.
b) Stain in alcian blue solution for 30 minutes.
c) Wash in running tap water for 2 minutes.
d) Rinse in distilled water.
e) Visualize under a light microscope and capture images for analysis. Blue
staining indicates synthesis of proteoglycans by chondrocytes.
TM
Fig.5 OriCell
Fischer 344(F344) Rat MSCs/RFP are differentiated into cartilages
and are stained with alcian blue.
IMPI0075A2 RAFMX‐01201 Page 11 of 15 CRYOPR
RESERVA
ATION OF
O CELL
LS USIN
NG OriCe
ellTM
CRYOPR
RESERVA
ATION MEDIA
M
R Protein-F
Free Cryoprreservation Medium (C
Cat. No. NC
CPF-10001) is a
OriCellTM NCR
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 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.
3.. Resuspen
nd the cell pellet in th e OriCellTM NCR Protein-Free Cry
yopreservation Medium
m
at a cell density
d
of 10
1 5-106 cellls/mL.
4.. Dispense
e aliquots of the cell su
uspension into cryogenic storage
e vials that are
properly labeled.
5.. Place the
e vials direc
ctly in a -80
0°C freezerr. After 24 hours, tran
nsfer the fro
ozen vials
to liquid nitrogen fo
or long-term
m preservattion.
IMPI0075A2 RA
AFMX‐01201 Page 12 of 15 APPENDIX
Troubleshooting
The table below lists some potential problems and solutions for culturing MSCs/RFP.
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.
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. Cell aging
IMPI0075A2 RAFMX‐01201 Page 13 of 15 Over digestion Cell aging
Wash the cells with PBS 2‐3 times to remove serum prior to trypsinization (serum will inhibit the function of trypsin).
Cells show
spontaneous
differentiation
Ineffective
induction of cell
differentiation
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
OriCellTM Mesenchymal Stem Cell Growth Medium
GUXMX-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
Jiang, Yuehua, Jahagirdar, Balkrishna N, and Reinhardt, R Lee.(2002)Pluripotency of
mesenchymal stem cells derived from adult marrow. Nature 418:41-49.
Hideya Yoshimura, Takeshi Muneta, and Akimoto Nimura. (2006)Comparison of rat
mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose
tissue, and muscle. Cell and Tissue Research 327:449-462.
IMPI0075A2 RAFMX‐01201 Page 14 of 15 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.
IMPI0075A2 RAFMX‐01201 Page 15 of 15