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HBEC-5i is a cerebral microvascular endothelial cell that was isolated from the cerebral cortex of a patient. This cell line was deposited by Kathryn Kellar, Ph.D. in 1994.
Product category
Human cells
Homo sapiens, human
Cell type
cerebral microvascular endothelial cell
Brain; Cerebral cortex
3D cell culture
Product format
Storage conditions
Vapor phase of liquid nitrogen
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ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain SV40 sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information


Specific applications
Studies in cancer, tumor metastasis, endothelium function, cell trafficking, surface molecule interactions, microvascular changes of the brain caused by malaria


HBEC-5i cells were derived from small fragments of human cerebral cortex obtained from patients who had died of various causes. These brains were devoid of any pathologic abnormalities. Isolation and purification procedures were performed and the cells were cultured. They were then transfected with plasmid containing SV40 large T antigen.
Immortalization method
SV40 large T antigen transformed
Antigen expression
The cells express von Willebrand's factor (vWF), VE-cadherin, occludin and cell adhesion molecules VCAM-1 and ICAM-1.
HBEC-5i (ATCC® No. CRL-3245™) has been shown to retain many of the characteristics of endothelial cells. These cells express stable patterns of endothelial cell markers such as VE-cadherin, von Willebrand factor VIII, and peripheral occludin, in addition to CD54, CD40, and CSA, and they show an up-regulation of CD54 and CD106 upon TNF activation. HBEC-5i cells exhibit major features of cerebral endothelial cells, especially efficient tight-junction structures, as assessed by high transendothelial electric resistance and very low permeability to 70-kDa dextran.These immortalized cells, because they are a continuously renewable source of human cerebral microvascular endothelial cells, can be used as a replacement for primary human brain endothelial cells for many research studies.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
The base medium for this cell line is DMEM:F12 (ATCC® 30-2006™). To make the complete growth medium, add the following components to the base medium: 

  • 40 µg/mL endothelial growth supplement (ECGS)
  • Fetal bovine serum (FBS; ATCC® 30-2020™) to a final concentration of 10%
Handling procedure
Note: These cells are cultured on vessels coated with 0.1% Gelatin (ATCC® No. PCS-999-027). Use 1.0 mL of gelatin per 10 cm2 surface area. Incubate at 37.0°C for ≥ 45 minutes. Aspirate gelatin just prior to adding cells to vessel(s). 

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –70°C. Storage at –70°C will result in loss of viability.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by
dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete growth medium and spin at approximately 125 x g for 5 to 7 minutes. Discard supernatant.
4. Resuspend the cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a gelatin-coated 25 cm2 or a 75 cm2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet
Subculturing procedure

Note: These cells are cultured on vessels coated with 0.1% Gelatin (ATCC® No. PCS-999-027). Use 1.0 mL of gelatin per 10 cm2 surface area. Incubate at 37.0°C for ≥ 45 minutes. Aspirate gelatin just prior to adding cells to vessel(s).

Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 
1. Remove and discard culture medium. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) (ATCC® No. 30-2200) or 0.25% Trypsin – 0.53mM EDTA (ATCC® No. 30-2101) solution to remove all traces of serum which contains trypsin inhibitor. 
2. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). 
     Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 
3. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting. 
4. Add appropriate aliquots of the cell suspension to new culture vessels. Incubate cultures at 37°C.

Subcultivation Ratio: 1:4 to 1:10 is recommended.
Medium renewal: 2 to 3 times per week
Reagents for cryopreservation
Complete growth medium supplemented with 7.5% (v/v) DMSO (ATCC 4-X)

Quality control specifications

STR profiling
TH01: 6,9.3
D5S818: 11,13
D13S317: 9,12
D7S820: 11
D16S539: 10,13
CSF1PO: 11,13
Amelogenin: X,Y
vWA: 14,18
TPOX:  8,11


Kathryn Kellar, Ph.D.
Year of origin

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.


This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.


Frequently Asked Questions


Curated Citations

Wassmer S, et al. TGF-B1 released from activated platelets can induce TNF-stimulated human brain endothelium apoptosis: a new mechanism for microvascular lesion during cerebral malaria. J. Immunol. 176: 1180-1184, 2006. PubMed: 16394007

Claessens A, et al. A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells. Proc. Natl. Acad. Sci. USA 109(26): E1772-E1781, 2012. PubMed: 22619330

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