Adipose-Derived Mesenchymal Stem Cells; Normal, Human

PCS-500-011 ^™

Normal Human Adipose-Derived Mesenchymal Stem Cells are fibroblast-like cells that, when grown in Mesenchymal Stem Cell Basal Media supplemented with Mesenchymal Stem Cell Growth Kit Low serum components, provide an ideal cell system to propagate mesenchymal stem cells in low serum (2% FBS) conditions. When maintained under optimal growth conditions, ATCC Normal Human Adipose-Derived Mesenchymal Stem Cells have been shown to be multipotent, capable of differentiating down the adipogenic, osteogenic, and chondrogenic lineages.
The cells are cryopreserved at the second passage to ensure the highest viability and plating efficiency. ATCC Primary Cell Solutions cells, media, supplements, and reagents are quality tested together to guarantee optimum performance and reliability.

Product category: Human cells
Organism: Homo sapiens, human
Cell type: mesenchymal stem cell
Morphology: Spindle-shaped, fibroblast-like
Tissue: Adipose tissue
Disease: Normal
Applications: Stem cell research

Cell and gene therapy (CGT) development
Product format: Frozen
Storage conditions: Vapor phase of liquid nitrogen

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Documentation

Product sheet

Characterization data

BSL 1

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

All tissues used for isolation are obtained under informed consent and conform to HIPAA regulations to protect the privacy of the donor’s Personally Identifiable Information. It is best to use caution when handling any human cells. We recommend that all human cells be accorded the same level of biosafety consideration as cells known to carry Human immunodeficiency virus (HIV) and other bloodborne pathogens. With infectious virus assays or viral antigen assays, even a negative test result may not exclude the possibility of the existence of a latent viral genome or infectious viral particles below the lower limit of detection of that assay.

ATCC recommends that appropriate safety procedures be used when handling all primary cells and cell lines, especially those derived from human or other primate material. Handle as a potentially biohazardous material using universal precautions. Cells derived from primate lymphoid tissue may fall under the regulations of 29 CFR 1910.1030 Bloodborne Pathogens.

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

Required Products

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

Mesenchymal Stem Cell Basal Medium for Adipose, Umbilical and Bone Marrow-derived MSCs

PCS-500-030

Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low Serum

PCS-500-040

Dulbecco's Phosphate Buffered Saline (D-PBS), 1X

30-2200

Trypsin-EDTA for Primary Cells

PCS-999-003

Trypsin Neutralizing Solution

PCS-999-004

Penicillin-Streptomycin-Amphotericin B Solution

PCS-999-002

Phenol Red

PCS-999-001

Adipocyte Differentiation Toolkit for Adipose-Derived MSCs and Preadipocytes

PCS-500-050

Chondrocyte Differentiation Tool

PCS-500-051

Osteocyte Differentiation Tool

PCS-500-052

General

Specific applications: Adult stem cell differentiation, regenerative medicine, cell therapy, tissue engineering, creation of iPS cell lines

Characteristics

Cells per vial: ≥ 1.0 x 10⁶
Volume: 1.0 mL
Growth properties: Adherent
Age: adult
Comments: The cells are cryopreserved in the second passage to ensure the highest viability and plating efficiency.

Mesenchymal Stem Cell Basal Medium, when supplemented with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low Serum (ATCC^® No. PCS-500-040) provides an ideal cell system to propagate mesenchymal stem cells in low serum (2% FBS) conditions. When maintained under optimal growth conditions, ATCC Normal Human Adipose-Derived Mesenchymal Stem Cells have been shown to be multipotent, capable of differentiating down the adipogenic, osteogenic and chondrogenic lineages.

Handling information

Unpacking and storage instructions

Check all containers for leakage or breakage.
Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below -130°C, preferably in liquid nitrogen vapor, until ready for use.

Complete medium

1. Obtain one Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low serum 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.
3. Obtain one bottle of Mesenchymal Stem Cell Basal Medium (485 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 indicated volume of each growth kit component, as indicated below, to the bottle of basal medium using a separate sterile pipette for each transfer.

Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low serum Components:

MSC Supplement, 10 mL, (2% FBS, 5 ng/mL rh FGF basic, 5 ng/mL rh FGF acidic, 5 ng/mL rh EGF);
L-Alanyl-L-Glutamine, 6 mL, 2.4 mM.

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 two weeks.

Reagents for subculture

D-PBS (ATCC 30-2200)
Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) containing 0.05% Trypsin and 0.02% EDTA. Note: Do not use other trypsin-EDTA concentrations with these primary cells.
Trypsin Neutralizing Solution (ATCC PCS-999-004)

Required media

One bottle of Mesenchymal Stem Cell Basal Medium (ATCC PCS-500-030) plus one Mesenchymal Stem Cell Growth Kit (ATCC PCS-500-040) that contains the following growth supplements: MSC Supplement (composed of FBS, rh FGF basic, rh FGF acidic, and rh EGF) and L-alanyl-L-glutamine

Optional media supplements

Gentamicin-Amphotericin B Solution (ATCC PCS-999-025)
Penicillin-Streptomycin-Amphotericin B Solution (ATCC PCS-999-002)
Phenol Red (ATCC PCS-999-001)

Handling procedure

Refer to the Certificate of Analysis for batch specific information on the total number of viable cells recovered from this lot of ATCC PCS-500-011.
Using the total number of viable cells, determine how much surface area can be inoculated to achieve an initial seeding density of 5,000 cells per cm².
Prepare the desired combination of flasks. Add 5 mL of complete growth medium 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.
While the culture flasks equilibrate, remove one vial of ATCC PCS-500-011 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).
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.
Add the appropriate volume of complete growth medium [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.
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.
Place the seeded culture flasks in the incubator at 37°C, 5% CO₂ atmosphere. Incubate for at least 24 hours before processing the cells further.

Subculturing procedure

Passage normal adipose-derived stem cells when the culture has reached approximately 70% to 80% confluence.
Warm both the Trypsin-EDTA for Primary Cells (ATCC^® No. PCS-999-003) and the Trypsin Neutralizing Solution (ATCC^® No. PCS-999-004) to room temperature prior to dissociation. Warm the complete growth medium to 37°C prior to use with the cells.
For each flask, carefully aspirate the spent media without disturbing the monolayer.
Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC^® No. 30-2200) to remove residual medium.
Add pre-warmed trypsin-EDTA solution (1 to 2 mL for every 25 cm²) to each flask.
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.
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.
When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin Neutralizing Solution (ATCC^® No. PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.
Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture flask.
Add 3 to 5 mL D-PBS (ATCC^® No. 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind.
Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA-dissociated cells.
Repeat steps 10 and 11 as needed until all cells have been collected from the flask.
Centrifuge the cells at 150 x g for 3 to 5 minutes.
Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm².
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

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.
24 hours after seeding, remove the cells from the incubator and view each flask under the microscope to determine percent cellular confluence.
Carefully remove the spent media without disturbing the monolayer.
Add 5 mL of fresh, pre-warmed complete growth medium per 25 cm² of surface area and return the flasks to the incubator.
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 70% to 80% confluence, and are actively proliferating (many mitotic figures are visible), it is time to subculture.

Note: Adipose-derived stem cells are contact inhibited. It is essential that the cells be subcultured BEFORE reaching confluence as post-confluent cells exhibit changes in morphology, slower proliferation, and reduced differentiation capacity after passaging.

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

Human immunodeficiency virus 2 (HIV-2): Not detected
Functional tests: Each lot of PCS-500-011 is tested to ensure in vitro differentiation to adipocytes.
Population doubling capacity: ≥ 10 in complete growth medium and support differentiation
Viability: ≥ 70% when thawed from cryopreservation
Specific staining: Positive for CD29, CD44, CD73, CD90, CD105, and CD166.
Negative for CD14, CD31, CD34, and CD45.

Legal disclaimers

Intended use

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

Warranty

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

Disclaimers

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

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

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

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

Permits & Restrictions

Import Permit for the State of Hawaii

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

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

Li Q, et al. Sulforaphane inhibits mammary adipogenesis by targeting adipose mesenchymal stem cells. Breast Cancer Res Treat 141(2):317-24, 2013. PubMed: 24002734

Tan KY, et al. Serum-free media formulations are cell line-specific and require optimization for microcarrierculture. Cytotherapy 17(8):1152-65, 2015. PubMed: 26139547

Sahin E, et al. Resveratrol reduces IL-6 and VEGF secretion from co-cultured A549 lung cancer cells and adipose-derived mesenchymal stem cells. Tumour Biol 37(6):7573-82, 2016. PubMed: 26687643

Gattu AK, et al. Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) resultsin increased adiposity and reduced bone mineral content. FASEB J 27(11):4384-94, 2013. PubMed: 23887690

Xu H, et al. Electrospun ultrafine fibrous wheat glutenin scaffolds with three-dimensionally randomorganization and water stability for soft tissue engineering. J Biotechnol 184:179-86, 2014. PubMed: 24862198

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