Human coronary artery smooth muscle cells人冠状动脉平滑肌细胞 (HCASMC) BioVector NTCC质粒载体菌种细胞基因保藏中心

Human coronary artery smooth muscle cells人冠状动脉平滑肌细胞 (HCASMC)

Primary Coronary Artery Smooth Muscle Cells; Normal, Human (HCASMC)
Cat No.: NTCC100021

NTCC® Normal Primary Coronary Artery Smooth Muscle Cells, when grown in Vascular Cell Basal Medium supplemented with Vascular Smooth Muscle Growth Kit components, provide an ideal cell system to propagate coronary artery smooth muscle cells in low serum conditions. The cells are cryopreserved in the second passage to ensure the highest viability and plating efficiency. NTCC® Primary Cell Solutions™ cells, media, supplements and reagents are quality-tested together to guarantee optimum performance and reliability.

Product category
Human cells
Product type
Primary cell
Organism
Homo sapiens, human
Cell type
myocyte
Morphology
Elongated and sometimes triangular in shape
Image:

Tissue
Coronary artery
Disease
Normal
Applications
3D cell culture
Cardiovascular disease research

General
Specific applications
Arteriolosclerosis, hypertension, arterial disease, organ-specific drug screening, vascular wound healing, tissue remodeling, vascular differentiation, apoptosis, stent development.
Characteristics
Cells per vial
≥ 5.0 x 105
Volume
1.0 mL
Growth properties
Adherent
Age
lot-specific
Ethnicity
Lot-specific
Gender
Lot-specific
Comments
The HCASMC cells are cryopreserved in the second passage to ensure the highest viability and plating efficiency.

A complete solution to propagate Vascular Smooth Muscle Cells in low serum conditions.
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 _x001f_-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
1. Obtain one growth kit from the freezer; make sure that the caps of all components are tight.
2. Thaw the components of the growth kit just prior to adding them to the basal medium. It is necessary to warm the L-glutamine component in a 37°C water bath and shake to dissolve any precipitates prior to adding to the basal medium.
3. Obtain one bottle of Vascular Cell Basal Medium (475 mL) from cold storage.
4. Decontaminate the external surfaces of all growth kit component vials and the basal medium bottle by spraying them with 70% ethanol.
5. Using aseptic technique, and working in a laminar flow hood or biosafety cabinet, transfer the volume of each growth kit component, as indicated below, to the bottle of basal medium using a separate sterile pipette for each transfer.

Vascular Smooth Muscle Cell Growth Kit Components:
rh FGF-basic, 0.5 mL, 5 ng/mL
rh Insulin, 0.5 mL, 5 µg/mL
Ascorbic acid, 0.5 mL, 50 µg/mL
L-glutamine, 25.0 mL, 10 mM
rh EGF, 0.5 mL, 5 ng/mL
Fetal Bovine Serum, 25.0 mL, 5%

Antimicrobials and phenol red are not required for proliferation but may be added if desired. The recommended volume of either of the optional components (GA solution or PSA solution) to be added to the complete growth media is summarized below.

Addition of Antimicrobials/Antimycotics and Phenol Red (Optional):
Gentamicin-Amphotericin B Solution, 0.5 mL, (Gentamicin: 10 µg/mL, Amphotericin B: 0.25 µg/mL)
Penicillin-Streptomycin-Amphotericin B Solution, 0.5 mL, (Penicillin: 10 Units/mL, Streptomycin: 10 µg/mL, Amphotericin B: 25 ng/mL)
Phenol Red, 0.5 mL, 33 µM

6. Tightly cap the bottle of complete growth medium and swirl the contents gently to assure a homogeneous solution. Do not shake forcefully to avoid foaming. Label and date the bottle.
7. Complete growth media should be stored in the dark at 2°C to 8°C (do not freeze). When stored under these conditions, complete growth media is stable for 30 days.
Reagents for subculture
1.D-PBS (NTCC 30-2200)
2.Trypsin-EDTA for Primary Cells (NTCC PCS-999-003) containing 0.05% Trypsin and 0.02% EDTA. Note: Do not use other trypsin-EDTA concentrations with NTCC PCS-100-021.
3.Trypsin Neutralizing Solution (NTCC PCS-999-004)
Required media
One bottle of Vascular Cell Basal Medium (NTCC PCS-100-030) plus one Vascular Smooth Muscle Cell Growth Kit (NTCC PCS-100-042) that contains the following components: recombinant human (rh) FGF-b, rh insulin, ascorbic acid, L-glutamine, rh EGF, and FBS.
Optional media supplements
1.Gentamicin-Amphotericin B Solution (NTCC PCS-999-025)
2.Penicillin-Streptomycin-Amphotericin B Solution (NTCC PCS-999-002)
3.Phenol Red (NTCC PCS-999-001)
Handling procedure
1.Refer to the batch specific information for the total number of viable cells recovered from this lot of NTCC® PCS-100-021.
2.Using the total number of viable cells, determine how much surface area can be inoculated to achieve an initial seeding density between 2,500 and 5,000 cells per cm2.
3.Prepare the desired combination of flasks. Add 5 mL of complete growth media per 25 cm2 of surface area. Place the flasks in a 37°C, 5% CO2, humidified incubator and allow the media to pre-equilibrate to temperature and pH for 30 minutes prior to adding cells.
4.While the culture flasks equilibrate, remove one vial of NTCC® PCS-100-021 from storage and thaw the cells 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 1 to 2 minutes).
5.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 onward should be carried out under strict aseptic conditions.
6.Add the appropriate volume of complete growth media [volume = (1 mL x number of flasks to be seeded) - 1 mL] into a sterile conical tube. Using a sterile pipette, transfer the cells from the cryovial to the conical tube. Gently pipette the cells to homogenize the suspension. Do not centrifuge.
7.Transfer 1.0 mL of the cell suspension to each of the pre-equilibrated culture flasks prepared in steps 1 to 3 of Handling Procedure for Frozen Cells and Initiation of Culture. Pipette several times, then cap and gently rock each flask to evenly distribute the cells.
8.Place the seeded culture flasks in the incubator at 37°C with a 5% CO2 atmosphere. Incubate for at least 24 hours before processing the cells further.
Subculturing procedure
1.Passage normal vascular smooth muscle cells when culture has reached approximately 80% confluence.
2.Warm both the Trypsin-EDTA for Primary Cells (NTCC PCS-999-003) and the Trypsin Neutralizing Solution (NTCC PCS-999-004) to room temperature prior to dissociation. Warm complete growth medium to 37°C prior to use with the cells.
3.For each flask, carefully aspirate the spent media without disturbing the monolayer.
4.Rinse the cell layer two times with 3 to 5 mL D-PBS (NTCC 30-2200) to remove residual traces of serum.
5.Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm2) to each flask.
6.Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.
7.Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 1 to 3 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.
8.When the majority of cells appear to have detached, quickly add an equal volume of Trypsin Neutralizing Solution (NTCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
9.Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the flask.
10.Add 3 to 5 mL D-PBS (NTCC 30-2200) to the flask to collect any additional cells that might have been left behind.
11.Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells.
12.Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
13.Centrifuge the cells at 150 x g for 3 to 5 minutes .
14.Aspirate the neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
15.Count the cells and seed new flasks at a density between 2,500 and 5,000 cells per cm2.
16.Place newly seeded flasks in a 37°C, 5% CO2, incubator for at least 24 to 48 hours before processing the cells further. Refer to Maintenance for guidelines on feeding.
Culture maintenance
1.Before beginning, pre-warm complete growth media in a 37°C water bath. This will take between 10 and 30 minutes, depending on the volume. If using a small volume of medium (50 mL or less), warm only the volume needed in a sterile conical tube. Avoid warming complete growth media multiple times.
2.24 to 36 hours after seeding, remove the cells from the incubator and view each flask under the microscope to determine percent cellular confluence.
3.Carefully remove the spent media without disturbing the monolayer.
4.Add 5 mL of fresh, pre-warmed complete growth media per 25 cm2 of surface area and return the flasks to the incubator.
5.After 24 to 48 hours, view each flask under the microscope to determine percent cellular confluence. If not ready to passage, repeat steps 3 and 4 as described above. When cultures have reached approximately 80% to 90% confluence, and are actively proliferating (many mitotic figures are visible), it is time to subculture. Vascular smooth muscle cells are contact inhibited; post-confluent vascular smooth muscle cells may not proliferate after passaging.
6.Note: Cells are typically ready to passage after 7 to 9 days in culture when inoculated with 2,500 cells/cm2.
Quality control specifications
Bacterial and fungal testing
Not detected
Mycoplasma contamination
Not detected
Virus testing
Human immunodeficiency virus 1 (HIV-1): Not detected
Hepatitis C virus (HCV): Not detected
Hepatitis B virus (HBV): Not detected
Population doubling capacity
≥ 15 in complete growth medium
Viability
≥ 70% when thawed from cryopreservation
Specific staining
Von Willebrand factor positive and smooth muscle alpha-actin negative.

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