人气管上皮细胞Cell Specification
The respiratory epithelium is composed of a mixed population of ciliated, nonciliated, and mucoussecreting
cells from proximal to distal airways. The individual characteristics of the subtypes of these
cells create not only an effective physical barrier against various noxious substances, but also a highly
sophisticated host defense system by producing and releasing a large number of chemical mediators and
cytokines [1]. The bronchial epithelium consists of the surface epithelial cells and mucus glands. The
surface epithelial cells are made up of three principle cell types: basal, goblet, and ciliated cells, of which
the latter two form a suprabasal columnar structure and are necessary for mucociliary clearance. Studies
using bronchial epithelial cell culture differentiated to induce a mucociliary phenotype have shown that
IL-4 and IL-13 stimulation modulate basic cell functions such as proliferation, ciliary beating, and
mucous production [2]. Cell proliferation, as well as various other cellular processes in bronchial
epithelial cells, is also regulated in part by EGF receptor signaling [3]. The availability of bronchial
epithelial cell culture provides an in vitro model to study the mechanisms of function and
pathophysiology of these cells.
HBEpiC from ScienCell Research Laboratories are isolated from human bronchi. HBEpiC are
cryopreserved at primary culture and delivered frozen. Each vial contains >5 x 105
cells in 1 ml volume.
HBEpiC are characterized by immunofluorescent method with antibodies CK-18, -19, and vimentin.
人气管上皮细胞HBEpiC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HBEpiC are
guaranteed to further culture at the conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Bronchial Epithelial Cell Medium (BECM, Cat. No. 3211) for the
culturing of HBEpiC in vitro.
Product Use
HBEpiC are for research use only. It is not approved for human or animal use, or for application
in in vitro diagnostic procedures.
Storage
Directly and immediately transfer cells from dry ice to liquid nitrogen upon receiving and keep
the cells in liquid nitrogen until cell culture needed for experiments.
Shipping
Dry ice.
Reference
[1]. Velden, V. H., and H. F. Versnel. 1998. Bronchial epithelium: morphology, function and pathophysiology in
asthma. Eur. Cytokine Netw. 9:585–597.
[2]. Kikuchi, T., Shively, J.D., Foley, J.S., Drazen, J.M., Tschumperlin, D.J. (2004) Differentiation-dependent
responsiveness of bronchial epithelial cells to IL-4/13 stimulation. Am J Physiol Lung Cell Mol Physiol.
287:L119-26.
[3]. Kim S, Schein AJ, Nadel JA.(2005) E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC
mucin expression in cultured human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol. 289:L1049-60.
Instruction for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C waterbath
and return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the ordering:
人气管上皮细胞1. Prepare a poly-L-lysine coated flask (2 μg/cm2
, T-75 flask is recommended). Add 10 ml
of sterile water to a T-75 flask and then add 15 μl of poly-L-lysine stock solution (10
mg/ml, ScienCell cat. no. 0413). Leave the flask in incubator overnight (minimum one
hour at 37o
C incubator).
2. Prepare complete medium: decontaminate the external surfaces of medium and medium
supplements with 70% ethanol and transfer them to sterile field. Aseptically open each
supplement tube and add them to the basal medium with a pipette. Rinse each tube with
medium to recover the entire volume.
3. Rinse the poly-L-lysine coated flask with sterile water twice and add 20 ml of complete
medium to the flask. Leave the flask in the hood and go to thaw the cells.
4. Place the vial in a 37o
C waterbath, hold and rotate the vial gently until the contents are
completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse
the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful
not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently
resuspend the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culture
vessels. A seeding density of 5,000 cells/cm2
is recommended.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of DMSO residue in the culture. It is
also important that HBEpiC are plated in poly-L-lysine coated flask that promote cell
attachment and growth.
6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen
cap if necessary to permit gas exchange.
7. Return the culture vessels to the incubator.
8. For best result, do not disturb the culture for at least 16 hours after the culture has been
initiated. Change the growth medium the next day to remove the residual DMSO and
unattached cells, then every other day thereafter. A health culture will display polygonal
shaped, sheets of contiguous cells and the cell number will be double after two to three
days in culture.
Maintenance of Culture:
1. Change the medium to fresh supplemented medium the next morning after establishing a
culture from cryopreserved cells. For subsequent subcultures, change medium 48 hours
after establishing the subculture.
人气管上皮细胞2. Change the medium every other day thereafter, until the culture is approximately 50%
confluent.
3. Once the culture reaches 50% confluence, change medium every day until the culture is
approximately 80% confluent.
Subculture:
1. Subculture the cells when they are over 90% confluent.
2. Prepare poly-L-lysine coated cell culture flasks (2 μg/cm2
).
3. Warm medium, trypsin/EDTA solution, trypsin neutralization solution, and DPBS to
room temperature. We do not recommend warming the reagents and medium at 37o
C
waterbath prior to use.
4. Rinse the cells with DPBS.
5. Incubate cells with 10 ml of trypsin/EDTA solution (in the case of T-75 flask) for around
5 min at 37o
C or until cells are completely rounded up (monitored with microscope).
Remove trypsin/EDTA solution and further incubate the cells at 37o
C for 1 min. With
one hand hold the flask, gently tap the edge of flask with the other hand to release cells
from the culture surface. Add 10 ml of trypsin neutralization solution to flask.
Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to
minimize the killing of the cells by over trypsinization.
6. Harvest and transfer released cells into a 50 ml centrifuge tube. Rinse the flask with
another 10 ml of growth medium to collect the residue cells. Examine the flask under
microscope to make sure the harvesting is successful by looking at the number of cells
left behind. There should be less than 5%.
7. Centrifuge the harvested cell suspension at 1000 rpm for 5 min and resuspend cells in
growth medium.
8. Count cells and plate them in a new, poly-L-lysine coated flask with cell density as
recommended.
Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative
for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions
mush be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working these materials.
人气管上皮细胞Never mouth pipette. We recommend following the universal procedures for handling products of human origin as
the minimum precaution against contamination [1].
[1]. Grizzle, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues. J Tissue Culture Methods. 11(4).