REF: P10951-IM

IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELL LINE

Product Type:

Immortalized Human Bladder Smooth Muscle Cell Line

Service Number:

P10951-IM

Source:Primary Smooth Muscle Cells from Human Bladder

Number of Cells:

1 x 106 Cells / vial (1ml)

Storage:

Liquid Nitrogen

P10561-IM have been obtained immortalizing

Recommended Medium

Human Bladder Smooth Muscle Cells with

Lenti-SV40 Lentivirus. Immortalized cells were

Smooth Muscle Cell Medium(Reference: P60125)

controlled passaging side by side with theprimary cells. Primary cells go into senescenceafter the 3rd passage while the SV40-tranduced
cells go beyond 30 passages.
The urinary bladder is a hollow organ made up
of smooth muscle cells. The relaxation andcontraction of urinary bladder smooth muscle
allows the bladder to store and void urine,respectively.
bladder

Phenotypicsmoothmusclemodulation

ofcells

theandexpression of inducible nitric oxide synthase
are associated with various pathologicalconditions, including bladder dysfunction. The
secretory phenotype of bladder smoothmuscle cell ECM can be altered by thefrequency

ofmechanical

Product CharacterizationImmunofluorescent methodoα‐smooth muscle actinoDesmin

The cells test negative for HIV‐1, HBV, HCV,
mycoplasma, bacteria, yeast and fungi

deformation

experienced by the cells. Since smooth

Product Usemuscle is the final common pathway for many

THESE PRODUCTS ARE FOR RESEARCH USE

diseases, to understand how smooth muscle

ONLY. Not approved for human or veterinarychanges during the genesis and maintenanceuse, for application to humans or animals, orof a disease is an important step toward thefor use in vitro diagnostic or clinicaldevelopment of therapeutic approaches.

procedures

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.
Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.innoprot.com

INSTRUCTIONS FOR CULTURING CELLSIMPORTANT: Cryopreserved cells are very delicate. Thaw the vial in a 37 ºC waterbathand return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
1.

2.

Prepare a poly‐L‐lysine‐coated culture
vessel (2 μg/cm2, T‐75 flask isrecommended). Add 10 ml of sterilewater to a T‐75 flask and then add 150
μl of poly‐L‐lysine stock solution (1mg/ml). Leave the vessel in a 37ºC
incubator overnight (or for aminimum of one hour).
Preparecompletemedium.
Decontaminate the external surfacesof medium bottle and medium
supplement tubes with 70% ethanol
and transfer them to a sterile field.
Aseptically transfer supplement tothe basal medium with a pipette.
Rinse the supplement tube withmedium to recover the entire
volume.

3. Rinse the poly‐L‐lysine‐coated vessel
twice with sterile water and then add
15 ml of complete medium. Leave thevessel in the sterile field and proceed
to thaw the cryopreserved cells.
4. Place the frozen vial in a 37ºC waterbath. Hold and rotate the vial gently
until the contents completely thaw.
Promptly remove the vial from thewater bath, wipe it down with 70%
ethanol, and transfer it to the sterilefield.
5. Carefully remove the cap without
touching the interior threads. Gently
resuspend and dispense the contentsof the vial into the equilibrated, poly‐
L‐lysine‐coated culture vessel. A
seeding density of 5,000 cells/cm2 isrecommended.

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.
Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.innoprot.com

Note: Dilution and centrifugation of cells
after thawing are not recommended
since these actions are more harmful
to the cells than the effect of residual
DMSO in the culture. It is also
important that cells are plated inpoly‐L‐lysine‐coated culture vessels
to promote cell attachment.
6. Replace the cap or lid of the culture
vessel and gently rock the vessel todistribute the cells evenly. Loosen
cap, if necessary, to allow gas
exchange.
7.

Return the culture vessel to theincubator.

8. For best results, do not disturb theculture for at least 16 hours after theculture has been initiated. Refresh
culture medium the next day toremove
residualDMSO
andunattached cells, then every otherday thereafter.

Maintenance of Culture:
1.

Refreshsupplementedculture
medium the next morning afterestablishing
aculture
fromcryopreserved cells.

2.

Change the medium every other day
thereafter,
until
theculture
is approximately 70% confluent.

3. Once the culture reaches 70%
confluency, change medium every
other day until the culture isapproximately 90% confluent.

Subculture:1.

Subculture when the culture reaches
90% confluency or above.

2.

Prepare poly‐L‐lysine‐coated culture
vessels (2 μg/cm2) one day before
subculture.

3. Warm
completemedium,trypsin/EDTA solution (T/E Solution),
T/E neutralization solution (TNS), andDPBS (Ca++‐ and Mg++‐free) to roomtemperature. We do not recommend
warming reagents and medium in a37ºC water bath prior to use.
Note: DPBS, trypsin/EDTA solution &trypsin neutralization solution are
included in the “Primary Cells Detach
Kit provided by Innoprot (Cat. NºP60305).

9. Add 5 ml of TNS solution to the flask
and transfer detached cells to the 50
ml centrifuge tube. Rinse the flaskwith another 5 ml of TNS to collect
the residual cells.
10. Examine the flask under amicroscope for a successful cell
harvest by looking at the number ofcells being left behind; there shouldbe less than 5%.
11. Centrifuge the 50 ml centrifuge tube
at 1000 rpm for 5 minutes.
Resuspend cells in culture medium.
12. Count and plate cells in a new poly‐L‐
lysine‐coated culture vessel with therecommended cell density.

4. Rinse the cells with DPBS.
5. Add 8 ml of DPBS and then 2 ml of T/E
solution into flask (in the case of a T‐
75 flask). Gently rock the flask toensure complete coverage of cells byT/E solution. Incubate the flask in a37ºC incubator for 1 to 2 minutes oruntil cells completely round up. Use amicroscope to monitor the change incell morphology.
6. During incubation, prepare a 50 ml
conical centrifuge tube with 5 ml offetal bovine serum (FBS).
7.

Transfer T/E solution from the flask to
the 50 ml centrifuge tube (a small
percent of cells may detach) andcontinue to incubate the flask at
37oC for another 1 to 2 minutes (nosolution in the flask at this moment).

8. At the end of incubation, gently tap
the side of the flask to dislodge cells
from the surface. Check under amicroscope to make sure that all cells
detach.

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.
Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.innoprot.com

Caution: Handling human derived products is
potentially bioharzadous. Although each cellstrain testes negative for HIV, HBV and HCVDNA, diagnostic tests are not necessarily 100%
accurate, therefore, proper precautions mushbe taken to avoid inadvertent exposure. Always
wear gloves and safety glasses when working
these materials. Never mouth pipette. We
recommend following the universal proceduresfor handling products of human origin as theminimum precaution against contamination [1].

[1]. Grizzle, W. E., and Polt, S. S. (1988)
Guidelines
toavoidpersonal
contamination by infective agents inresearch laboratories that use humantissues. J Tissue Culture Methods.
11(4).