人毛囊角质细胞Cell Specification
Hair follicles are self-renewing structures that cycle and regenerate throughout life and also
provide a critical niche for keratinocyte stem cells [1]. The interplay between keratinocytes and
the underlying mesenchyme are critical for hair follicle morphogenesis and cycling. Several key
developmental pathways are involved in determining follicular keratinocyte fate as well as
the differentiation into several distinct cellular layers of the follicle during the growth phase of
hair [2]. Studies indicate that follicular keratinocytes are distinct from epidermal keratinocytes in
proliferative capacity and CD34 expression [3].
HHFK from ScienCell Research Laboratories are isolated from the human scalp. HHFK are
cryopreserved at P0 and delivered frozen. Each vial contains >5 x 105
cells in 1 ml volume.
HHFK are characterized by immunofluorescence with antibody specific to cytokeratin. HHFK
are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. HHFK are
guaranteed to further expand for 15 population doublings under the conditions provided by
ScienCell Research Laboratories.
Recommended Medium
It is recommended to use Keratinocyte Medium (KM, Cat. #2101) for culturing HHFK in vitro.
Product Use
HHFK are for research use only. They are not approved for human or animal use, or for
application in in vitro diagnostic procedures.
Storage
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and
keep the cells in liquid nitrogen until they are needed for experiments.
Shipping
Dry ice.
References
人毛囊角质细胞[1] Ohyama M, Terunuma A, Tock CL, Radonovich MF, Pise-Masison CA, Hopping SB, Brady JN, Udey MC,
Vogel JC. (2006) “Characterization and isolation of stem cell-enriched human hair follicle bulge cells.” J Clin
Invest. 116: 249-60.
[2] Mecklenburg L, Tychsen B, Paus R. (2005) “Learning from nudity: lessons from the nude phenotype.” Exp
Dermatol. 14: 797-810.
[3] Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM, Tennant RW. (2003) “Enrichment
for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34.” J Invest Dermatol.
120: 501-11.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC water bath
and return the cells to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare a poly-L-lysine-coated culture vessel (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, Cat. #0413). Leave the vessel in a 37oC incubator overnight (or for a
minimum of one hour).
人毛囊角质细胞2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field.
Aseptically transfer supplement to the basal medium with a pipette. Rinse the supplement
tube with medium 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 the vessel in the sterile field and proceed to thaw the
cryopreserved cells.
4. Place the frozen vial in a 37oC water bath. Hold and rotate the vial gently until the
contents completely thaw. Promptly remove the vial from the water bath, wipe it down
with 70% ethanol, and transfer it to the sterile field.
5. Carefully remove the cap without touching the interior threads. Gently resuspend and
dispense the contents of the vial into the equilibrated, poly-L-lysine-coated culture vessel.
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 residual DMSO in the culture. It is
also important that cells are plated in poly lysine coated culture vessels to promote cell
attachment.
6. Replace the cap or lid of the culture vessel and gently rock the vessel to distribute the
cells evenly. Loosen cap, if necessary, to allow gas exchange.
7. Return the culture vessel to the incubator.
8. For best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Refresh culture medium the next day to remove residual DMSO and unattached
cells, then every other day thereafter.
Maintaining the culture:
1. Refresh supplemented culture medium the next morning after establishing a culture from
cryopreserved cells.
2. Change the medium every three days thereafter, until the culture is approximately 70%
confluent.
3. Once the culture reaches 70% confluency, change medium every other day until the
culture is approximately 90% confluent.
Subculturing:
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 complete medium, trypsin/EDTA solution (T/E, Cat. #0103), T/E neutralization
solution (TNS, Cat. #0113), and DPBS (Ca++
- and Mg++
-free, Cat. #0303) to room
temperature. We do not recommend warming reagents and medium in a 37oC water bath
prior to use.
4. Rinse the cells with DPBS.
5. Add 5 ml of DPBS and then 5 ml of T/E solution into flask (in the case of a T-75 flask).
Gently rock the flask to ensure complete coverage of cells by T/E solution. Incubate the
flask in a 37oC incubator for 3 to 5 minutes or until cells completely round up. Use a
microscope to monitor the change in cell morphology.
6. During incubation, prepare a 50 ml conical centrifuge tube with 5 ml of fetal bovine
serum (FBS, Cat. #0500).
人毛囊角质细胞7. Transfer T/E solution from the flask to the 50 ml centrifuge tube (a small percent of cells
may detach) and continue to incubate the flask at 37oC for another 1 to 2 minutes (no
solution 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 a microscope to make sure that all cells detach.
9. Add 5 ml of TNS solution to the flask and transfer detached cells to the 50 ml centrifuge
tube. Rinse the flask with another 5 ml of TNS to collect the residual cells.
10. Examine the flask under a microscope for a successful cell harvest by looking at the
number of cells being left behind; there should be less than 5%.
Note: Use ScienCell T/E solution that is optimized to minimize cell damages due to over
trypsinization.
人毛囊角质细胞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 the recommended
cell density.
Caution: Handling human derived products is potentially biohazardous. Although each cell
strain tests negative for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100%
accurate, therefore, proper precautions must be taken to avoid inadvertent exposure. Always
wear gloves and safety glasses when working with these materials. Never mouth pipette. We
recommend following the universal procedures for handling products of human origin as the
minimum precaution against contamination [1].
[1] Grizzle WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues.” J Tissue Cult Methods. 11: 191‐9.