ATCC hTERT-immortalized Cell Culture Guide

Get tips and techniques for culturing hTERT-immortalized cells and ensure that your research is reproducible.

Table of Contents

Telomeres and Telomerase
ATCC hTERT-immortalized Cell Solutions
ATCC hTERT-immortalized Barrett’s Esophageal Epithelial Cells
ATCC hTERT-immortalized Bronchial Epithelial Cells
ATCC hTERT-immortalized Chondrocyte Fibroblast Cells
ATCC hTERT-immortalized Dermal Microvascular Endothelial Cells
ATCC hTERT-immortalized Endometrial Fibroblasts
ATCC hTERT-immortalized Human Foreskin Fibroblasts
ATCC hTERT-immortalized Mammary Epithelial Cells
ATCC hTERT-immortalized Pancreas Duct Epithelial Cells
ATCC hTERT-immortalized Renal Epithelial Cells
ATCC hTERT-immortalized Retinal Pigmented Epithelial Cells
Appendix
Tissue Types
References

Telomeres are repetitive DNA sequences that stabilize the terminal ends of the chromosomes. During each cell division, 50-200 base pairs of DNA are lost from the telomere ends of the chromosomes, and chromosomal shortening eventually leads to replicative senescence.¹ Telomerase is an enzyme, comprising an RNA component (ie, hTERC or hTR) and a catalytic component (ie, hTERT), that is able to restore the DNA base pairs lost from the telomeres during cell division. In cells with active telomerase, chromosomal length is maintained and the cells continue to divide without becoming senescent.¹

The RNA component of telomerase is expressed ubiquitously², while the expression of the catalytic hTERT component is mainly limited to the early stages of embryonic development, during which time it is expressed by stem cells. In the adult, its expression is restricted to some rare cells of the blood (ie, white blood cells), germ cells, and some cells of the skin and digestive track. Since most normal somatic cells do not have active telomerase, these cells are susceptible to replicative senescence in vivo and are difficult to maintain in vitro.¹

hTERT immortalization of primary cells

Transfection of hTERT into human primary cells leads to elongation and maintenance of the telomere ends of the chromosomes. In many instances, forced expression of hTERT alone enables the cells to repress replicative senescence and overcome the growth crisis, effectively leading to their immortalization.^2,3,4 In some cases, more than one immortalization agent may be required to successfully immortalize a particular cell type. For example primary cell lines may be immortalized using a combination of hTERT with one or more of the following: genes encoding viral [simian virus 40 (SV40) large T antigen^5,6 and human papilloma virus-16 (HPV-16) E6/E7^7,8] or non-viral (Cdk-4⁹ and Bmi-1¹⁰) oncoproteins.

Figure 1. Eukaryotic expression plasmid containing the hTERT reverse transcriptase cDNA.¹⁶

hTERT-immortalized cells are mostly diploid, but may become pseudo-diploid especially at high passage number. In many cases, when cells become pseudo-diploid they still retain most primary cell functions (Table 1). The following is a list of primary human cells that have been established by the forced expression of hTERT alone.

• Endothelial cells¹²
• Esophageal squamous cells²
• Fibroblasts¹¹
• Keratinocytes¹⁴
• Mammary epithelial cells¹³
• Nestin-positive cells of the pancreas¹⁶
• Osteoblasts¹⁵
• Retinal pigmented epithelial cells³

Properties of hTERT-immortalized cells

Early-passage hTERT-immortalized cells faithfully represent the physiological properties of normal cells in vivo. Additionally, analysis of numerous hTERT-immortalized cell lines has confirmed that these cells retain the expression of phenotypic markers and have a stable karyotype.^13,14 This is in contrast to many traditional lines of immortalized cells, which are known to develop an unstable karyotype, especially at high passage numbers and when oncogenes are used. Below is a summary of the commonly observed properties of cells immortalized by the ectopic expression of hTERT.

• Nonmalignant^17,19
• Normal cell cycle controls, functional p53 and pRB checkpoints^17,18
• Contact inhibited¹⁹
• Anchorage dependent¹⁹
• Retain normal growth responses to serum and mitogens³
• Require growth factors for proliferation³
• Possess a normal karyotype^4,18,*
• Do not show changes associated with transformation such as tumorigenicity or growth in soft agar²⁰

Benefits of hTERT-immortalized cells

Primary cells closely represent the physiological state of a particular cell type in vivo, but they are susceptible to replicative senescence, so their value in the laboratory setting is limited. This is especially true when large quantities of cells are required for biochemical analysis, genetic manipulations or for genetic screens. It is also a factor for the study of some types of rare hereditary human diseases, since the volume of the biological samples collected (biopsies or blood) is usually small and contains a limited number of cells. Continuous cell lines, on the other hand, are not encumbered by replicative senescence, but, they often contain numerous genetic mutations, exhibit an unstable karyotype and have protein expression patterns that are not comparable with the cell type they are intended to represent.

hTERT cells combine the physiological attributes of primary cell lines and the long culture life of continuous cell lines, while avoiding the replicative senescence of the former and the unstable karyotype of the latter. Additionally, in many studies hTERT-immortalized cells have been induced to become differentiated cell types, exhibiting tissue-specific features, differentiation-specific proteins, and forming structures that resemble those formed in vivo.¹⁷

Table 1. Comparison between hTERT-immortalized cells, primary cells, oncogene/viral-immortalized cells, and continuous cell lines

Applications

The unique properties of hTERT-immortalized cells, as discussed above (and illustrated in Table 1), make them an attractive replacement for both primary and transformed cell lines and a valuable tool for the study of cell functions both in vitro and in vivo. The following is a list of published applications of hTERT-immortalized cells.

• Long-term studies of biochemical and physiological aspects of cell growth, eg, endogenous protein markers, gene expression, and growth inhibition¹²
• In vitro model for differentiation and carcinogenesis^7,12
• Cancer research and studies of oncogenes^7,25
• Cell-based drug screening, and drug toxicity testing²⁶
• Tissue engineering, and transplantation²⁷
• Genetic engineering and modifications²⁸
• Biological functions of hTERT²⁹

*For karyotype information please see appendix.

ATCC hTERT-immortalized cell lines represent a breakthrough in cell biology research that combine the in vivo nature of primary cells and the in vitro utility of continuous cell lines. Until recently, cell biologists had to choose between primary cells and established cell lines as the basis for their experimental models, but both options had their flaws. Normal primary cells are difficult to isolate, often vary from lot to lot, and senesce after a few passages. Traditional cell lines, on the other hand, are genetically unstable and present inconsistent phenotypes over time. Now, with hTERT-immortalized cell lines from ATCC, cell biologists can avoid the limitations, while enjoying the benefits of both.

In addition to the standard ATCC authentication, all ATCC hTERT cell lines are tested for:

• Extended proliferative capacity
• Stable karyotype
• Selected phenotypic markers from the tissue of interest
• Continued expression of hTERT

ATCC hTERT-cell immortalization tools

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence, caused by the shortening of telomeres that occurs each time somatic human cells divide.¹ The enzyme telomerase can prevent the shortening of telomeres, and the transfer of exogenous hTERT cDNA (encoding the catalytic subunit of human telomerase) can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells.

ATCC hTERT-immortalization vectors

This set of immortalization products enables researchers to immortalize primary cells of interest in their labs using the hTERT technology.

• hTERT vectors: ATCC MBA-141 
• Viral oncoprotein vectors:
  • HPV-16 E6/E7: ATCC 45113; ATCC 45113D 
• Non-viral (Cdk-4 and Bmi-1) oncoprotein vectors: 81582D

Introduction

Primary Barrett’s epithelial cells have a limited lifespan in culture and typically do not contain the genetic abnormalities that can lead to cancer in Barrett’s esophagus, which significantly limits their use as a model for the progression of this disease. In contrast, ATCC hTERT-immortalized Barrett’s esophageal epithelial cells contain stable, defined cell cycle and genetic abnormalities, have an extended life span, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells.

The Barrett’s esophagus cell lines, CP-A (KR-42421), ATCC CRL-4027, CP-B (CP-52731), ATCC CRL-4028, CP-C (CP-94251), ATCC CRL-4029, and CP-D (CP-18821), ATCC CRL-4030 were derived from an endoscopic biopsy specimen obtained from a region of non-dysplastic metaplasia and transduced with the retroviral expression vector, pLXSN-hTERT.²

Cell culture protocols

ATCC CRL-4027 stained with a monoclonal pan-cytokeratin antibody (green) and Hoechst dye (blue)

Materials needed

• MCDB-153 medium (Sigma, M7403)
• Hydrocortisone
• Recombinant human epidermal growth factor
• Cholera toxin
• Adenine
• Bovine pituitary extract
• Insulin-transferrin-sodium selenite supplement (Sigma, I1884)
• Glutamine
• Fetal bovine serum (ATCC 30-2020) 
• Dulbecco’s phosphate buffered saline (ATCC 30-2200) 
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101) 
• Cell culture tested DMSO (ATCC 4-X) 
• RPMI-1640 medium (ATCC 30-2001) 

Preparation of complete growth medium

The base medium for these cell lines is MCDB-153. To make the complete growth medium add the following components to the base medium:

• 0.4 μg/mL hydrocortisone
• 20 ng/mL recombinant human epidermal growth factor
• 1 nM cholera toxin
• 20 mg/L adenine
• 140 μg/mL bovine pituitary extract
• 0.1% insulin-transferrin-sodium selenite supplement
• 4 mM glutamine
• 5% fetal bovine serum

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC primary Barrett’s epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Determine the number of viable cells and add an appropriate aliquot of the suspension to a culture flask.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium required proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. To facilitate dispersal of cells place the flask at 37°C.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. Proceed to the next step when the cells have reached the recommended density and are ready to be subcultured.
3. Remove and discard the media.
4. Rinse the cells with Dulbecco’s phosphate buffered saline to remove any trace of serum.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
9. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: RPMI-1640 Medium supplemented with 10% fetal bovine serum and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

The use of normal and diseased primary bronchial epithelial cultures is restricted by the limited availability of normal and diseased primary cell pairs, significant inconsistency between donors and their finite proliferative capacity. Moreover, the available cell lines have been transformed using viral genes or derived from tumors and do not maintain the morphology and phenotype of the parent cells. However, ATCC hTERT-immortalized bronchial epithelial cells have an extended lifespan, have a stable karyotype, and are phenotypically similar to the primary parent cells.

The human airway epithelial cell line, NuLi-1 (ATCC CRL- 4011), was derived from normal lung epithelial cells by dual retroviral infection with HPV-16 E6/E7-LXSN and hTERT-LXSN. Human airway epithelial cell lines, CuFi-1 (ATCC CRL-4013), CuFi-4 (ATCC CRL-4015), CuFi-5 (ATCC CRL-4016), and CuFi-6 (ATCC CRL-4017) were derived from the lung epithelial cells of cystic fibrosis patients by dual retroviral infection with HPV-16 E6/E7-LXSN and hTERT-LXSN or pBabe-hygro-hTERT.⁹

Cell culture protocols

ATCC CRL-4011 stained with a monoclonal pan-cytokeratin antibody (green) and Hoechst dye (blue)

Materials needed

• Bronchial Epithelial Growth Medium (BEGM), serum-free (Lonza BEGM BulletKit, CC-3170)
• G-418
• Human placental collagen type IV, (Sigma, C-7521)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101) 
• Cell culture tested DMSO (ATCC 4-X) 
• Fetal bovine serum (ATCC 30-2020) 
• Dulbecco’s phosphate buffered saline (ATCC 30-2200) 

Preparation of complete growth medium

The base medium for these cell lines is BEBM. To make the complete growth medium (BEGM) add the following components to the base medium:

• SingleQuot additives (supplied with the BEGM BulletKit)
• 50 µg/mL G-418

Note:
ATCC does not use the gentamycin-amphotericin B supplied with the BEGM kit.

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.

The culture flasks should be pre-coated with 60 µg/mL solution of human placental collagen type IV at least 18 hours in advance, then air-dried and rinsed 2 to 3 times with Dulbecco’s phosphate buffered saline.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC primary bronchial epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing procedure

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
The culture flasks should be pre-coated with 60 µg/mL solution of human placental collagen type IV at least 18 hours in advance, then air-dried and rinsed 2 to 3 times with Dulbecco’s phosphate buffered saline.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days (do not exceed 3 days).
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Add 2.0 to 3.0 mL trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
5. To remove trypsin-EDTA solution, add 2.0 to 3.0 mL of 1% FBS in Dulbecco’s phosphate buffered saline and aspirate cells by gently pipetting.
6. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
7. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
8. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

Cryopreservation medium

Freeze cells in the following medium: BEGM supplemented with 10% DMSO and 30% fetal bovine serum. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

Primary human fetal chondrocytes exhibit a relatively short life span under standard culture conditions, which severely restricts their ability to be used in research studies. ATCC hTERT-immortalized chondrocytes have an extended lifespan, retain expression of chondrocyte specific markers, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells.

The chondrocyte cell lines, CHON-001 (ATCC CRL-2846) and CHON-002 (ATCC CRL-2847), were derived from chondrocytes of normal human long bones infected by hTERT-LXSN under G-418 selection.

Cell culture protocols

Materials needed

• ATCC-formulated Dulbecco’s modified Eagle’s medium (DMEM) (ATCC 30-2002)
• G-418
• Fetal bovine serum (ATCC 30-2020)
• Dulbecco’s phosphate buffered saline (ATCC 30-2200)
• Trypsin-EDTA Solution (0.05% Trypsin/0.53 mM EDTA, such as Gibco 25300-054) or Trypsin-EDTA for primary cells (ATCC PCS-999-003)
• Cell culture tested DMSO (ATCC 4-X)

Preparation of complete growth medium

The base medium for these cell lines is ATCC-formulated DMEM. To make the complete growth medium, add the following components to the base medium:

• 0.1 mg/mL G-418
• 10% heat-inactivated fetal bovine serum

Note:
This medium is formulated for use with a 5% CO₂ in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO₂ in air atmosphere is then recommended).

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC primary human fetal chondrocytes.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing procedure

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 days, or as needed.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Rinse the cells with Dulbecco’s phosphate buffered saline to remove traces of serum.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. To remove trypsin-EDTA solution, add 2.0 to 3.0 mL of 1% FBS in Dulbecco’s phosphate buffered saline and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 90% heat-inactivated fetal bovine serum and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

ATCC hTERT-immortalized dermal microvascular endothelial cells have an extended lifespan, express a panel of endothelial cell surface proteins, undergo tubule formation in culture, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells. By overcoming the finite proliferative capacity of primary cells, the hTERT dermal microvascular endothelial cells represent an effective cell model for studying endothelial cell biology including signal transduction and angiogenesis.

The telomerase-immortalized human microvascular endothelium cell line, TIME (ATCC CRL-4025), was derived from a primary culture of neonatal foreskin microvascular endothelial cells (HMVEC) of the dermis. Primary HMVECs were immortalized by infection with the retrovirus WZLblast3:hTERT. TIME cells express a panel of characteristic endothelial cell surface marker proteins including CD31/PECAM-1 and integrin αVβ3. The cells also express the low density lipoprotein (LDL) receptor and are capable of acetylated LDL uptake.¹²

Cell culture protocols

ATCC CRL-4025 stained with a CD31 antibody (green) and Hoechst dye (blue)

Materials needed

• Endothelial Cell Basal Medium-2 (EBM-2), (Lonza EGM-2-MV BulletKit, CC-3202)
• Blasticidine
• Trypsin-EDTA solution (Lonza, CC-5012)
• Trypsin neutralizing solution (Lonza, CC-5002)
• Fetal bovine serum (ATCC 30-2020)
• HEPES buffered saline solution (Lonza, CC-5024)
• Cell culture tested DMSO (ATCC 4-X)

Preparation of complete growth medium

The base medium for these cell lines is Endothelial Cell Basal Medium-2 (EBM-2). To make the complete growth medium (EGM-2), add the following components to the base medium:

• SingleQuot additives supplied with the kit 
• 12.5 µg/mL blasticidine

Note:
ATCC does not use the gentamycin-amphotericin B mix supplied with the kit.

Note:
Do not filter complete medium.

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and not at -70°C. Storage at –70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC dermal microvascular endothelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing procedure

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made, proceed to the next step.
3. Remove and discard the media.
4. Rinse the cells with room temperature HEPES buffered saline solution.
5. Add 5.0 to 6.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 4 to 8 minutes).
6. To inactivate trypsin-EDTA solution, add 5.0 to 6.0 mL of trypsin neutralizing solution and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 90% fetal bovine serum and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

ATCC hTERT-immortalized endometrial fibroblasts (T HESCs) have an extended lifespan and are karyotypically, morphologically, and phenotypically similar to the primary parent cells. Functionally, T HESCs display the biochemical endpoints of decidualization after hormone treatment.

T HESCs (ATCC CRL-4003) were derived from stromal cells obtained from an adult female with myomas. The primary stromal endometrium cells were immortalized by infection with supernatant from the packaging cell line pA317-hTERT which expressed the hTERT and the puromycin resistance genes.²³

Cell culture protocols

Materials needed

• Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Sigma, D2906)
• Sodium bicarbonate
• ITS+ Universal Culture Supplement Premix (BD, 354352)
• Puromycin
• Charcoal/dextran treated fetal bovine serum (Hyclone, SH30068.03)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101)
• Cell culture tested DMSO (ATCC 4-X)

Preparation of complete growth medium

The base medium for these cell lines is DMEM/F12 supplemented with:

• 1.5 g/L sodium bicarbonate
• 1% ITS+ Universal Culture Supplement Premix
• 500 ng/mL puromycin
• 10% charcoal/dextran treated fetal bovine serum

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC endometrial fibroblasts cells.
2. Prepare a 25 cm² or a 75 cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 6.0 to 8.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing procedure

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not hit or shake the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made, proceed to the next step.
3. Remove and discard the media.
4. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).
5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
6. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
7. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
   Refer to the product sheet for the recommended inoculum concentration.

Cryopreservation medium

Freeze cells in the following medium: 95% complete growth media, 5% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

Human foreskin fibroblasts were among the first primary cell types to be successfully immortalized using hTERT technology, so the functions and properties of hTERT-immortalized fibroblasts have been thoroughly characterized. ATCC hTERT-immortalized foreskin fibroblasts (BJ-5ta) have an extended lifespan, express PDGFRβ, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells. BJ 5ta are widely used as in vitro models for wound healing, tissue engineering, and regeneration applications.

The hTERT-immortalized foreskin fibroblast cell line, BJ-5ta (ATCC CRL-4001), was derived by transfecting the BJ foreskin fibroblast cell line, at population doubling (PDL) 58, with pGRN145 hTERT followed by selection with hygromycin B.²⁵

Cell culture protocols

Materials needed

• Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC 30-2002)
• M199 medium
• Hygromycin B
• Fetal bovine serum
• Dulbecco’s phosphate buffered saline (ATCC 30-2200)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101)

Preparation of complete growth medium

The base medium for this cell line is a 4:1 mixture of DMEM and M199. To make the complete growth medium add the following components to the base medium:

• 0.01 mg/mL hygromycin B
• 10% fetal bovine serum

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of human foreskin fibroblasts.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Rinse the cells with Dulbecco’s phosphate buffered saline to remove traces of serum.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate the cultures in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 90% fetal bovine serum and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

ATCC hTERT-immortalized mammary epithelial cells (hTERT-HME1) have an extended lifespan, do not express estrogen receptor, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells. In addition, these cells retain many differentiated features of normal HMECs. As such, hTERT-HME1 can serve as valuable in vitro models to study the stages of breast cancer development.

The human mammary epithelium, HME1 (ATCC CRL-4010) cell line was derived from normal primary mammary epithelial cells infected with the retrovirus pBabepuro+hTERT vector and cultured in complete growth medium containing puromycin until stable clones were selected.¹³

Cell culture protocols

ATCC CRL-4010 stained with a monoclonal pan-cytokeratin antibody (green) and Hoechst dye (blue)

Materials needed

• Mammary Epithelial Growth Medium, serum-free (MEGM) from Clonetics (Lonza MEGM BulletKit, CC-3150)
• Soybean trypsin inhibitor (ATCC 30-2104)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101)
• Dulbecco’s phosphate buffered saline (ATCC 30-2200)

Preparation of complete growth medium

The base medium for these cell lines is MEBM. To make the complete growth medium (MEGM) add the following components to the base medium:

• SingleQuot additives (supplied with the MEGM BulletKit)

Note:
ATCC does not use the gentamycin- amphotericin B mix provided with the kit. Do not filter complete medium.

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC immortalized mammary epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Add 2.0 to 3.0 mL of soybean trypsin inhibitor solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
6. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
7. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.

Cryopreservation medium

Freeze cells in the following medium: 90% complete culture medium supplemented and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

Investigating the roles played by cells expressing the neuronal stem cell marker nestin in the human pancreas is important in pancreatic cancer and metabolic disease studies. ATCC hTERT-immortalized pancreas duct epithelial cells have an extended lifespan, express nestin, and are karyotypically, morphologically, and phenotypically similar to the primary parent cells.

hTERT-HPNE (Human Pancreatic Nestin Expressing cells) (ATCC CRL-4023) was developed from human pancreatic duct cells by transduction with a retroviral expression vector (pBABEpuro) containing the hTERT gene. hTERT-HPNE E6/E7 (ATCC CRL- 4036) cells were derived from hTERT-HPNE cells (ATCC CRL- 4023) by infection with retroviral vector pLEXSN carrying HPV-16 E6/E7. hTERT-HPNE E6/E7/st cells were derived from hTERT-HPNE E6/E7 cells (ATCC CRL-4036) by infection with retroviral vector pBabeZeo carrying the SV40 small T-antigen. hTERT-HPNE E6/E7/K-RasG12D cells were derived from hTERT-HPNE E6/E7 cells (ATCC CRL-4036) by infection with retroviral vector pLXSN carrying a G12D mutant of the isoform b of human K-Ras. hTERT-HPNE E6/E7/K-RasG12D/st cells were derived from hTERT-HPNE E6/E7/K-RasG12D cells (ATCC CRL-4038) by infection with retroviral vector pBabeZeo carrying the SV40 small T-antigen.⁷

Cell culture protocols

ATCC CRL-4023 stained with a monoclonal pan-cytokeratin antibody (red) and Hoechst dye (blue)

Materials needed

• DMEM without glucose (Sigma D-5030 with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate)
• Medium M3 Base (Incell Corp. M300F-500)
• Recombinant human epidermal growth factor
• D-glucose
• Puromycin
• Cell culture tested DMSO (ATCC 4-X)
• Fetal bovine serum (ATCC 30-2020)
• Dulbecco’s phosphate buffered saline (ATCC 30-2200)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101)

Preparation of complete growth medium

The base medium for this cell line is:

• 75% DMEM without glucose
• 25% Medium M3 Base

To make the complete growth medium, add the following components to the base medium:

• Fetal bovine serum to a final concentration of 5%
• 10 ng/mL human recombinant EGF
• 5.5 mM D-glucose (1g/L)
• 750 ng/mL puromycin

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC pancreas duct epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Rinse the cells with Dulbecco’s phosphate buffered saline.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate cultures in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 90% fetal bovine serum and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

Angiomyolipomas are benign tumors of the kidney, which originate from putative perivascular epithelioid cells. These cells may undergo differentiation into cells with features of melanocytes, smooth muscle, or fat cells. However, the study of angiomyolipomas is limited by a lack of established angiomyolipoma-derived cell lines and a lack of good animal models. ATCC hTERT-immortalized renal epithelial cells are derived from angiomyolipomas, so researchers can now take the advantage of both the primary cell characteristics and extended lifespan of these cells in vitro.

The UMB1949 cell line (ATCC CRL-4004) expresses NG2 and L1 and has a defined 5bp deletion in exon 33 of tuberin (Tsc2) and mutations in tuberin (and/or hamartin). As such, this cell line can be used to study signal transduction and drug efficiency in tuberous sclerosis. The SV7tert PDGFtu1 (ATCC CRL-4008) cell line is derived from the tumors caused by the SV7tert implantation in nude mice. The SV7tert cell line is a non-tumorigenic angiomyolipoma cell line immortalized with the SV40 large T antigen and human telomerase, by transduction with a retrovirus encoding PDGF-BB. The tumor-derived cells secrete over 18-fold more PDGF than pre-implantation cells, and demonstrate both autocrine transformation and epigenetic changes.⁵

Cell culture protocols

ATCC CRL-4004 stained with a monoclonal pan-cytokeratin antibody (green) and Hoechst dye (blue)

Materials needed

• Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC 30-2002)
• Fetal bovine serum (ATCC 30-2020)
• Dulbecco’s phosphate buffered saline (ATCC 30-2200)
• Trypsin-EDTA Solution (0.25% Trypsin/0.53 mM EDTA in HBSS) (ATCC 30-2101)
• Cell culture tested DMSO (ATCC 4-X)

Preparation of complete growth medium

The base medium for these cell lines is DMEM. To make the complete growth medium add the following components to the base medium:

• Fetal bovine serum to a final concentration of 10%

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC renal epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Rinse the cells with Dulbecco’s Phosphate Buffered Saline to remove traces of serum.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate the cultures in a 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 95% complete growth medium, 5% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

Introduction

Retinal pigment epithelium (RPE) is the layer of cells at the base of the retina composed of a single layer of hexagonal cells that are densely packed with pigment granules. The RPE protects and supplies nutrition to the retina, and degeneration of the RPE is linked to age-related macular degeneration (AMD). The hTERT-immortalized RPE cell line is a valuable tool in AMD research, with application to studies of RPE functions, disease progression, RPE regeneration, and RPE wound healing.

The hTERT-immortalized retinal pigmented epithelial cell line hTERT RPE-1 (ATCC CRL-4000) was derived by transfecting the RPE-340 cell line with the pGRN145 hTERT-expressing plasmid (ATCC MBA-141). Cells were cultured in medium containing hygromycin B until stable clones were selected.¹¹

Cell culture protocols

ATCC CRL-4000 stained with a monoclonal pan-cytokeratin antibody (green) and Hoechst dye (blue)

Materials needed

• Dulbecco’s Modified Eagle’s Medium (DMEM)/ F12 (ATCC 30-2006)
• Fetal bovine serum (ATCC 30-2020)
• Hygromycin B
• Hank's balanced salt solution (ATCC 30-2213)
• Trypsin-EDTA Solution (ATCC 30-2101)
• Cell culture tested DMSO (ATCC 4-X)

Preparation of complete growth medium

The base medium for this cell line is ATCC-formulated DMEM: F12. To make the complete growth medium, add the following components to the base medium:

• Fetal bovine serum to a final concentration of 10%
• 0.01 mg/mL hygromycin B

Handling procedure for frozen cells and initiation of cultures

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor and not at -70°C. Storage at -70°C will result in loss of viability.

1. Refer to the batch specific information provided on the last page of the product information sheet for the total number of viable cells recovered from each lot of ATCC retinal pigmented epithelial cells.
2. Prepare a culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
3. 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).
4. 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 operations from this point on should be carried out under strict aseptic conditions.
5. Transfer the vial contents to a centrifuge tube containing 9.0 mL of complete culture medium and centrifuge the cell suspension at approximately 125 x g for 5 to 7 minutes.
6. Discard the supernatant and resuspend the cells in fresh growth medium (see the batch-specific information for the recommended dilution ratio). Add this suspension to the prepared culture vessel.
7. Incubate the culture in a 37°C, 5% CO₂, humidified incubator.

Subculturing and maintenance of cultures

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note:
To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Place at 37°C to facilitate dispersal.

1. Maintain the cells in culture by refreshing the medium every 2 to 3 days.
2. Refer to the product information sheet for the concentration at which cells should be subcultured. When a determination that the cells are ready to be subcultured is made proceed to the next step.
3. Remove and discard the media.
4. Rinse with Hank's balanced salt solution to remove traces of serum.
5. Add 2.0 to 3.0 mL of trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 10 minutes).
6. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
7. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
8. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. Refer to the product sheet for the recommended inoculum concentration.
9. Incubate cultures at 37°C, 5% CO₂, humidified incubator.

Cryopreservation medium

Freeze cells in the following medium: 30% culture medium, 60% fetal bovine serum, and 10% DMSO. Store vials in liquid nitrogen vapor. Avoid immersing vials into liquid nitrogen.

hTERT karyotype information

Applications

1. Long-term studies of biochemical and physiological aspects of cell growth; 2. In vitro model for differentiation and carcinogenesis; 3. Cancer research and studies of oncogenes; 4. Genetic engineering and modifications; 5. Tissue engineering, and transplantation; 6. Gene expression studies; 7. Cell-based drug screening, and drug toxicity testing; 8. Biological functions of the hTERT

For information about depositor, immortalization method, growth medium, karyotype, and references please consult the appropriate product detail page.

1. Shay JW, Wright WE. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 2005; 26: 867-874.
2. Palanca-Wessels MC, Barrett MT, Galipeau PC, Rohrer KL, Reid BJ, Rabinovitch PS. Genetic analysis of long-term Barrett’s esophagus epithelial cultures exhibiting cytogenetic and ploidy abnormalities. Gastroenterology 1998; 114: 295-304.
3. Rambhatla L, Chiu CP, Glickman RD, Rowe-Rendleman C. In vitro differentiation capacity of telomerase immortalized human RPE cells. Invest Ophthalmol Vis Sci 2002; 43: 1622-1630.
4. Arbiser JL, Yeung R, Weiss SW, Arbiser ZK, Amin MB, Cohen C, Frank D, Mahajan S, Herron GS, Yang J, Onda H, Zhang HB, Bai X, Uhlmann E, Loehr A, Northrup H, Au P, Davis I, Fisher DE, Gutmann DH. The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms. Am J Pathol 2001; 159: 483- 491.
5. Lim SD, Stallcup W, Lefkove B, Govindarajan B, Au KS, Northrup H, Lang D, Fisher DE, Patel A, Amin MB, Arbiser JL. Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells? Mol Med 2007; 13: 160-165.
6. Campbell PM, Groehler AL, Lee KM, Ouellette MM, Khazak V, Der CJ. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res 2007; 67: 2098-2106.
7. Zabrenetzky V, et al. In: Annual meeting of the ASCB; 1997.
8. Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, Peyton M, Zou Y, Kurie JM, Dimaio JM, Milchgrub S, Smith AL, Souza RF, Gilbey L, Zhang X, Gandia K, Vaughan MB, Wright WE, Gazdar AF, Shay JW, Minna JD. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 2004; 64: 9027-9034.
9. Haga K, Ohno S, Yugawa T, Narisawa-Saito M, Fujita M, Sakamoto M, Galloway DA, Kiyono T. Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT. Cancer Sci 2007; 98: 147-154.
10. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE. Extension of life-span by introduction of telomerase into normal human cells. Science 1998; 279: 349-352.
11. Venetsanakos E, Mirza A, Fanton C, Romanov SR, Tlsty T, McMahon M. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells. Exp Cell Res 2002; 273: 21-33.
12. Herbert BS, Wright WE, Shay JW. p16(INK4a) inactivation is not required to immortalize human mammary epithelial cells. Oncogene 2002; 21: 7897-7900.
13. Harada H, Nakagawa H, Oyama K, Takaoka M, Andl CD, Jacobmeier B, von Werder A, Enders GH, Opitz OG, Rustgi AK. Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation. Mol Cancer Res 2003; 1: 729-738.
14. Thi MM, Urban-Maldonado M, Spray DC, Suadicani SO. Characterization of hTERT-immortalized osteoblast cell lines generated from wild-type and connexin43-null mouse calvaria. Am J Physiol Cell Physiol; 299: C994-C1006.
15. Lee KM, Nguyen C, Ulrich AB, Pour PM, Ouellette MM. Immortalization with telomerase of the Nestin- positive cells of the human pancreas. Biochem Biophys Res Commun 2003; 301: 1038-1044.
16. Li H, Zhou J, Miki J, Furusato B, Gu Y, Srivastava S, McLeod DG, Vogel JC, Rhim JS. Telomerase- immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells. Exp Cell Res 2008; 314: 92-102.
17. Toouli CD, Huschtscha LI, Neumann AA, Noble JR, Colgin LM, Hukku B, Reddel RR. Comparison of human mammary epithelial cells immortalized by simian virus 40 T-Antigen or by the telomerase catalytic subunit. Oncogene 2002; 21: 128-139.
18. Jaiswal KR, Morales CP, Feagins LA, Gandia KG, Zhang X, Zhang HY, Hormi-Carver K, Shen Y, Elder F, Ramirez RD, Sarosi GA, Jr., Spechler SJ, Souza RF. Characterization of telomerase-immortalized, non-neoplastic, human Barrett’s cell line (BAR-T). Dis Esophagus 2007; 20: 256-264.
19. Dalerba P, Guiducci C, Poliani PL, Cifola I, Parenza M, Frattini M, Gallino G, Carnevali I, Di Giulio I, Andreola S, Lombardo C, Rivoltini L, Schweighoffer T, Belli F, Colombo MP, Parmiani G, Castelli C. Reconstitution of human telomerase reverse transcriptase expression rescues colorectal carcinoma cells from in vitro senescence: evidence against immortality as a constitutive trait of tumor cells. Cancer Res 2005; 65: 2321-2329.
20. Delgado O, Kaisani AA, Spinola M, Xie XJ, Batten KG, Minna JD, Wright WE, Shay JW. Multipotent capacity of immortalized human bronchial epithelial cells. PLoS One; 6: e22023.
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