In Vitro Technologies

Permits and Restrictions	View Permits
Organism	Homo sapiens, human
Tissue	Brain
Cell Type	Neural
Product Format	frozen
Morphology	Neurosphere
Culture Properties	Suspension
Biosafety Level	1 [It is the responsibility of the investigator to determine appropriate safety procedures for use with this material. As a reference, laboratory safety is discussed in the publication Biosafety in Microbiological and Biomedical Laboratories and can be accessed by searching "BMBL" at www.cdc.gov.] Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
Disease	Oligoastrocytoma Grade III
Age	38 years
Gender	Male
Ethnicity	Caucasian
Storage Conditions	Liquid Nitrogen Vapor Phase

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Clinical Data	38 years Male Caucasian Oligoastrocytoma Grade III
Comments	Point mutations in isocitrate dehydrogenase I (IDH1) and IDH2 are found in majority of grade II and III gliomas. R132H is the most common IDH1 substitution found in gliomas. BT142 mut/- contains a homozygous IDH1 R132H mutation, which originated from a heterozygous IDH1 R132H BT142 cells. The cells grow as phase-bright, smooth spheres. The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells. The cells should be passaged when the neurospheres are 200 to 400 µm in size.

Images

Clinical Data

38 years Male Caucasian Oligoastrocytoma Grade III

Comments

Point mutations in isocitrate dehydrogenase I (IDH1) and IDH2 are found in majority of grade II and III gliomas. R132H is the most common IDH1 substitution found in gliomas.

BT142 mut/- contains a homozygous IDH1 R132H mutation, which originated from a heterozygous IDH1 R132H BT142 cells.

The cells grow as phase-bright, smooth spheres.

The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells.

The cells should be passaged when the neurospheres are 200 to 400 µm in size.

Complete Growth Medium	There are two options for the base medium for this cell line. Option 1: NeuroCult NS-A Proliferation kit (Catalog No. 5751, Stem Cell Technologies) Option 2: DMEM/F12 (1:1) (Catalog No. 30-2006, ATCC) with an additional 0.9% glucose, 4 mM L-glutamine (Catalog No. 30-2214, ATCC), 25 µg/mL insulin, 100 µg/mL transferrin, 20 nM progesterone, 15 µM putrescine and 30 nM selenite To make the complete growth medium, add the following supplements to either options of the base medium (see above): 20ng/mL recombinant human Epidermal Growth Factor (EGF, Catalog No. 100-15, PeproTech) 100 ng/mL recombinant human Platelet-Derived Growth Factor-AA (PDGF-AA, Catalog No. 100-13A, PeproTech) 20 ng/mL recombinant human Fibroblast Growth Factor (R&D Systems, Catalog No. 233-FB) 2 µg/mL heparan sulfate (Catalog No. H3149, Sigma)
Subculturing	The cells grow as phase-bright, smooth spheres. The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells. The cells should be passaged when the neurospheres are about 200-400 μm in size. Volumes used in this protocol are for 75 cm² flask. Harvest and collect the entire cell suspension from the culture flask into a 15 mL tube. Centrifuge at 200 x g for 10 minutes. Aspirate supernatant, leaving approximately 200 µL to cover the pellet. Add 1 mL of complete culture medium. Triturate cells with a P1000 micropipette set to 800 µL by pipetting up and down 40 times or until the cells appear to be in a single cell suspension. Add 8 mL of complete culture medium and centrifuge at 200 x g for 10 minutes. Aspirate supernatant and resuspend the cells in 2 mL of complete culture medium. Count viable cells using trypan blue exclusion assay on a hemacytometer. Seed single cells between ranges of 8 x 10³ to 2 x 10⁴ cells/cm². Note: If accurate cell count is necessary, Accumax (Catalog No. AM105, Innovative Cell Technologies) can be used; however, the cells may take some time to recover from an enzymatic dissociation.
Cryopreservation	Freeze medium: Complete growth medium (90%) supplemented with 10% (v/v) DMSOStorage temperature: liquid nitrogen vapor phase

Complete Growth Medium

There are two options for the base medium for this cell line. Option 1: NeuroCult NS-A Proliferation kit (Catalog No. 5751, Stem Cell Technologies) Option 2: DMEM/F12 (1:1) (Catalog No. 30-2006, ATCC) with an additional 0.9% glucose, 4 mM L-glutamine (Catalog No. 30-2214, ATCC), 25 µg/mL insulin, 100 µg/mL transferrin, 20 nM progesterone, 15 µM putrescine and 30 nM selenite To make the complete growth medium, add the following supplements to either options of the base medium (see above):

20ng/mL recombinant human Epidermal Growth Factor (EGF, Catalog No. 100-15, PeproTech)
100 ng/mL recombinant human Platelet-Derived Growth Factor-AA (PDGF-AA, Catalog No. 100-13A, PeproTech)
20 ng/mL recombinant human Fibroblast Growth Factor (R&D Systems, Catalog No. 233-FB)
2 µg/mL heparan sulfate (Catalog No. H3149, Sigma)

Subculturing

The cells grow as phase-bright, smooth spheres. The neurospheres should not get too big, ragged or dark as this is a sign of unhealthy, dying cells. The cells should be passaged when the neurospheres are about 200-400 μm in size.

Volumes used in this protocol are for 75 cm² flask.

Harvest and collect the entire cell suspension from the culture flask into a 15 mL tube.
Centrifuge at 200 x g for 10 minutes.
Aspirate supernatant, leaving approximately 200 µL to cover the pellet.
Add 1 mL of complete culture medium.
Triturate cells with a P1000 micropipette set to 800 µL by pipetting up and down 40 times or until the cells appear to be in a single cell suspension.
Add 8 mL of complete culture medium and centrifuge at 200 x g for 10 minutes.
Aspirate supernatant and resuspend the cells in 2 mL of complete culture medium.
Count viable cells using trypan blue exclusion assay on a hemacytometer.
Seed single cells between ranges of 8 x 10³ to 2 x 10⁴ cells/cm².

Note: If accurate cell count is necessary, Accumax (Catalog No. AM105, Innovative Cell Technologies) can be used; however, the cells may take some time to recover from an enzymatic dissociation.

Cryopreservation

Freeze medium: Complete growth medium (90%) supplemented with 10% (v/v) DMSOStorage temperature: liquid nitrogen vapor phase

Name of Depositor	This cell line was deposited by Samuel Weiss, Ph.D. and Gregory Cairncross, Ph.D. of the University of Calgary
References	Luchman HA et al. An in vivo patient-derived model of endogenous IDH1-mutant glioma. Neuro Oncol. 14:184-191, 2012. PubMed: 22166263

E: care@invitro.com.au
P: 1300 552 003

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BT142 oligoastrocytoma, Grade III Item ID: ATCACS1018

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