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Immortalized Human Tonsillar Epithelial Cells (HTE)

Cat. No.

T0721

Unit

1x10⁶ cells / 1.0 ml

Price

$610.00

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Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing institution.

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Cat. No.	T0721
Name	Immortalized Human Tonsillar Epithelial Cells (HTE)
Description	Immortalized Human Tonsillar Epithelial (HTE) cells are isolated from healthy adult patient biopsy and immortalized by being transfected with human papillomavirus type 16 (HPV16). HTE cells can be a useful cell line model as HPV-transfected human tonsillar epithelial cells for studying the HPV pathology and the role of HPV in the etiology of HPV related cancer. HTE cells can also be used as a cell model to study human tonsillar epithelial cell physiology and function.
Organism	Human (H. sapiens)
Tissue	Tonsil
Donor History	Not disclosed
Growth Properties	Adherent, polygonal
Cell Type	Immortalized Cells
Unit	1x10⁶ cells / 1.0 ml
Storage Condition	Vapor phase of liquid nitrogen, or below -130°C.
Shipping Conditions	Ship with dry ice.
Product Format	Frozen
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.
BioSafety	II
Certificate of Analysis	For batch-specific test results, refer to the applicable certificate of analysis that can be found at www.abmgood.com.
Growth Conditions	Use of PriCoat^TM T25 Flasks (G299) is required for cell adhesion to the culture vessels. Prigrow X Series Medium for T0721 (TM0721) + 200 ug/ml G418 (G271) for selection pressure, 37.0°C, 5% CO₂. Change media every 2-3 days once co-culture has been established. Cells must be co-cultured with mitomycin C-treated mouse NIH 3T3 J2 fibroblasts as a feeder layer. Refer to the "Thawing Protocol" and "Subculturing Protocol" sections for detailed instructions on cell handling.
Unpacking and Storage Instructions	1. Visually examine the packaging containers for signs of leakage or breakage. 2. Immediately transfer frozen cells from dry ice packaging to a temperature below -130°C, preferably in liquid nitrogen vapor phase storage, until ready for use. To ensure the highest level of viability, thaw the vial and initiate culture as soon as possible upon receipt. If continued storage is desired, the vial should only be stored below -130°C or in liquid nitrogen vapor phase. Do not store at -70°C, as it will result in loss of viability.
Thawing Protocol	Feeder cells (NIH 3T3 J2 fibroblasts) must be prepared prior to thawing T0721. To prepare the feeder cell layer: Thaw NIH 3T3 J2 fibroblasts as per supplier instructions. Once fibroblasts reach 40-50% confluency, add Mitomycin C (15 ug/mL) and incubate at 37°C for three hours. Remove mitomycin C containg growth media and wash cells for a total of 5X using PBS (1X) to remove any residual traces of mitomycin C. Add fresh growth media to the culture vessel and incubate at 37°C (5% CO₂) for 1-2 hours to allow cells to adapt. Note: feeder cells may change morphology post-treatment with mitomycin C and some floating cells may be observed. Although cells may appear bigger in size and present multiple nuclei, confluency should not increase post-treatment. The feeder lay is now ready for T0721. Prepare T0721 for co-culture: Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination. Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions. Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes. Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media. Co-culture T0721 and NIH 3T3 J2 fibroblasts feeder cells: Aspirate feeder cell growth media from the culture vessel containing NIH 3T3 J2 fibroblasts feeder cells and replace with T0721 complete growth media. Add the re-suspended T0721 cells (from step 4 above) to the culture vessel and incubate cells according to the recommended culture conditions. T0721 may take additional time to recover from post-thaw conditions, therefore, allow cells to attach over the next 24-48 hours before changing media. A successful co-culture will present growing colonies of T0721 between the feeder cells. T0721 cells may grow on-top of one another.
Subculture Protocol	Volumes given below are for a T75 flask; proportionally increase or decrease the volume as required per culture vessel size. Subculture cells once the culture vessel is 80% confluent. Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel. Incubate at 37°C for one minute to remove feeder cell layer. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes) of only the feeder cells. Collect the suspended feeder cells and discard. Add 2-3ml of fresh, pre-warmed 0.25% Trypsin-EDTA to the culture vessel. Incubate at 37°C for 2-3 minutes. Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media to the culture vessel. Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type. Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels that have a feeder cell layer ready. Incubate the cells at the recommended conditions.
Cryopreservation	Cryopreservation Medium (TM024), or complete growth media with 10% DMSO.
Immortalization Method	Dual infection with E6 and E7 HPV16 viral genes.
Expression	E7 and E1^E4 expression; G418-resistant
Warranty	abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period”.
Disclaimer	1. Sale of this item is subjected to the completion of a Material Transfer Agreement (MTA) by the purchasing individual/institution for each cell line. If you have any questions regarding this, please contact us at licensing@abmgood.com. 2. All test parameters provided in the CoA are conducted using abm's standardized culture system and The stated values may vary under the end-user's culture conditions. Please verify that the product is suitable for your studies by referencing published papers or ordering RNA (0.5 μg, Cat.# C207, $450.00) or cell lysate (100 μg, Cat.# C206, $600.00) to perform preliminary experiments, or alternatively use our Gene Expression Assay Service (Cat# C138). All sales are final. 3. We recommend live cell shipments for ease of cell transfer and this option can be requested at the time of order placement. Please note that the end-user will need to evaluate the feasibility of live cell shipment by taking into account the final destination's temperature variation and its geographical location. 4. All of abm's cell biology products are for research use ONLY and NOT for therapeutic/diagnostic applications. abm is not liable for any repercussions arising from the use of its cell biology product(s) in therapeutic/diagnostic or any other non-RUO application(s). 5. abm makes no warranties or representations as to the accuracy of the information on this site. Citations from literature are provided for informational purposes only. abm does not warrant that such information has been shown to be accurate. 6. abm warrants that cell lines shall be viable upon initiation of culture for a period of thirty (30) days after shipment and that they shall meet the specifications on the applicable abm Material Product Information sheet, certificate of analysis, and/or catalog description. Such thirty (30) day period is referred to herein as the "Warranty Period".
Depositor	LSU Health Sciences Center - Shreveport
Application	Research Use Only.
Material Citation	If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. T0721

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Supporting Protocol

Other

	I want to make sure these cells express my gene of interest before I decide to buy the cell line. Can you provide a sample so this can be tested?
	We do not perform extensive downstream characterization, or gene expression profiling of our cell lines. However, you can check the ‘References’ tab to see if a publication is available, or you can contact us to see if RNA or cell lysates are available for your cell line of interest. Please directly contact us for further information at quotes@abmgood.com.

	Does abm's PriGrow series medium contain antibiotics?
	No, the medium does not contain antibiotics and needs to be supplemented by the end-user as desired.

	Do I need to use abm's media and Applied Cell Extracellular Matrix (G422) to culture my cells?
	We strongly encourage using the recommended media and culture conditions described in the "Growth Conditions" section as they have been optimized for cell growth. Other supplier's media and coating conditions have not been tested.

	What is the difference between Applied Cell Extracellular Matrix (G422) and Collagen Coating?
	The main component of our Applied Cell Extracellular Matrix (G422) is type I collagen specifically. For more information on abm's Applied Cell Extracellular Matrix please visit: http://www.abmgood.com/Applied-Cell-Extracellular-Matrix-G422.html.

	Do I have to use T25 ECM-coated flasks for growing the cells?
	We strongly recommend thawing cells in the T25 flasks specified on the cell-specific product page to ensure optimal post-thaw recovery of the frozen cells before testing other culture vessels.

	If I want to plate these cells to multi-well plates (e.g. 96 well plates) or dishes, how should I prepare the plates?
	For a protocol on how to coat plates and dishes with Applied Cell Extracellular Matrix (Cat. No. G422), please download the “Applied Cell Extracellular Matrix Data Sheet” from here.

	What percentage of Trypsin-EDTA should be used to subculture cells? Can I use Trypsin-EDTA containing phenol red?
	The standard concentration used for subculturing is 0.25% Trypsin-EDTA, available at abm (Cat. No. TM050). Yes, Trypsin-EDTA containing phenol red can be used as it does not interfere with trypsinization.

	How often do I need to change the media?
	Media should be changed every 2-3 days or as specified within the recommended growth conditions.

Why are these cells classified as biosafety level II?

We follow the CDC-NIH recommendations that all mammalian sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products. This information can be found in 'Biosafety in Microbiological and Biomedical Laboratories' (1999). Your institution's Safety Officer or Technical Services will be able to make the call as to whether BioSafety Level I is possible with these cells at your site, if desired.

	How long can I store frozen vials?
	Cells that are properly frozen using an effective cryoprotective agent can be stored in liquid nitrogen vapor phase (or below -130°C) indefinitely without affecting viability.

What is the recommended storage temperature for cells?

In general, if you received:

Live cells: acclimatize for 3-5 hours at the recommended temperature and CO2 conditions stated for the cell line under the Growth Conditions section, and then change media afterwards. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).
Frozen cells: Upon receipt, immediately place cells in liquid nitrogen; or below -130°C.

	Can I substitute heat-inactivated FBS with FBS or vice versa?
	FBS and heat-inactivated FBS are different in their composition; they cannot be substituted for one another.

My cells are not detaching, what method do you recommend to trypsinize the cells?

Gently wash adherent cells with PBS (1X, without calcium or magnesium) to remove trypsin inhibitors before adding the trypsinization agent.
Ensure the correct trypsin concentration is being used (i.e. 0.25%).
Do not use cold trypsin to detach cells as the proteolytic activity will be low. Pre-warm trypsin to 37°C for optimal enzymatic activity.
Incubate the cells with trypsin-edta agent at 37°C to facilitate cell detachment.
Gently tap the side of the culture vessel to facilitate mechanical detachment.

How are live cells shipped?

Live cells are shipped as a live culture in a T12.5 flask. Cells are seeded at an optimal confluency to avoid metabolic waste build up during transport. In addition, we thoroughly test our cell lines for functional freeze-thaw recovery. For more information on how to handle live cells, please view our detailed instructions here (https://www.abmgood.com/uploads/document/Cell_Handling_Instructions_Upon_Arrival_150623.pdf).

	Why are cells not attaching and forming clumps after subculturing with trypsin?
	Cells form clumps and display attachment issues when the trypsinization agent is not effectively neutralized. To completely neutralize the trypsin agent, add an equal amount of complete growth media prior to centrifugation. Subtle changes in culture conditions, particularly in pH and the quality of serum used in the growth medium, may also affect the amount of clumping exhibited by the cells.

	Why are my cells forming clumps after plating?
	It is important to ensure cell clumps are broken up through gentle re-suspension by pipetting up and down several times after pelleting cells by centrifugation. This will ensure a homogenous single cell suspension for even plating.

	Do I need to add the selection drug to the complete growth medium?
	We recommend maintaining selection pressure using the drug concentration specified in the Growth Conditions section.

	Where can I find the CoA for my product?
	Certificates of analysis can be found here (https://www.abmgood.com/CoA-Library.html).

Spanos, W. C., Geiger, J., Anderson, M. E., Harris, G. F., Bossler, A. D., Smith, R. B., Klingelhutz, A. J., & Lee, J. H. (2008). Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells. Head & neck, 30(2), 139–147. https://doi.org/10.1002/hed.20673

Bienkowska-Haba, M., Luszczek, W., Myers, J. E., Keiffer, T. R., DiGiuseppe, S., Polk, P., Bodily, J. M., Scott, R. S., & Sapp, M. (2018). A new cell culture model to genetically dissect the complete human papillomavirus life cycle. PLoS pathogens, 14(3), e1006846. https://doi.org/10.1371/journal.ppat.1006846

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