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Biotech-Catalog Cell Biology/Cell Banking-Storage |
Cell Biology Testing - Cell Banking | AppTec offers a variety of standard and custom GLP-compliant cell culture services for our clients. Our Cell Biology department can provide cell growth services to supply the cells needed for multiple tests, as well as design and implement custom cell-based assays to specific client requirements. In addition, our established bank of cell lines contains a wide variety of human, animal, insect and other eukaryotic cell lines that can be used in viral detection or other assays. Well-characterized cell banks exist for all cell lines in order to produce cells of the highest quality cells for use in biosafety testing. Each cell bank is fully tested – according to the most rigorous, up-to-date test methods – to show identity and whether any contaminating adventitious infectious agents or retroviral particles are present. Fully tested cell lines most commonly used and readily available for your testing needs include those listed below. Additional cell lines are cryopreserved and available upon request. HUMAN | 324-K
A 549 | BJAB
CEM-A | H9
HEK 293 | HeLa
HeLa-S3 | HEL 299
Hs 68 | HT-1080
MRC-5 | RD
WI-38 |
MURINE | Balb/3T3
K-BALB | L-929
Mus dunni | NIH/3T3
SC-1 |
| | MONKEY | FRhK-4
VERO | CV-1
LLC-MK2 | BS-C-1
MA 104 |
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| | Cell Line Expansion & Characterization Back to Top |
| 30040 | This procedure is designed to optimize the production of test article cells and derivative materials in the shortest possible time for subsequent use in sponsor-designated assays. Test article materials are generated for: 1) protocols using
cell-free supernatants from cultures in exponential growth, 2) protocols using 107 or fewer cells, and 3) protocols using more than 107 cells.
Request Protocol 30184 to order cell growth for assays that includes small-scale cryopreservation. The cell line is cryopreserved at 3 x 106 to 1 x 107 cells/vial using a slow freezing regimen.
Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks which are filled almost to the top with medium, capped tightly and sealed with Parafilmâ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on the Sample Submission Form, as well as sufficient growth medium and supplements for at least two weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), and media components. Also list any drugs or special additives present in media. | Cell Growth for Assays
Turnaround Time:
2-4 weeks
(Dependent on cell growth and number of tests ordered) Container/Volume:
2 x 1 ml cryopreserved cells or
5 x 106 viable cells Shipment Temperature:
Cryopreserved cells: On dry ice
Viable cells: Ambient |
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| 30117 | The appearance and growth characteristics of a cell line under defined culture conditions are described. The determination of cellular morphology includes: cell type, cell shape, two-dimensional cell size, and general appearance. Growth is characterized by the number of cell doublings within a given period of time and cell density at saturation. Photomicrographs of representative phase and differential interference contrast microscopy are included.
Transmission electron microscopy can be performed to visualize sub-cellular components (Protocol 30257), to detect and quantitate virus particles (Protocol 30023), or both (Protocol 30610). This will add three (3) weeks to the turnaround time. Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks which are filled almost to the top with medium, capped tightly and sealed with Parafilmâ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on the Sample Submission Form, as well as sufficient growth medium and supplements for at least two weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), and media components. Also list any drugs or special additives present in media. | | Determination of Cellular Morphology and Growth Characteristics
Turnaround Time:
4 weeks Container/Volume:
2 x 1 ml cryopreserved cells or
5 x 106 to 1 x 107 viable cells Shipment Temperature:
Cryopreserved cells: On dry ice
Viable cells: Ambient |
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| 30006 | This procedure determines the ability of test article cells to form colonies in soft agar. This test takes advantage of the fact that normal cells are anchorage-dependent, while transformed cells are anchorage-independent. This property can be demonstrated by the formation of colonies in a semisolid medium. Cells are cultured in a semisolid medium for two weeks. Microscopic examination of colony formation and photomicrographs of selected fields are made after the first and second weeks. The positive control for this test is a human fibrosarcoma (HT-1080) cell line. The negative control cell line is diploid human lung (WI-38). Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks which are filled almost to the top with medium, capped tightly and sealed with ParafilmÒ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on Sample Submission Form, as well as sufficient growth medium and supplements for at least two weeks in culture.
·Provide S.O.P. or protocols for growth conditions (+/- trypsin), and media components. Also list any drugs or special additives present in media.
· If cells are submitted cryopreserved, Protocol 30040 (Cell Growth for Assays) should be performed. | In Vitro Tumorigenicity: Colony Formation in Soft Agar
Turnaround Time:
4 weeks Container/Volume:
5 x 106 to 1 x 107 viable cells Shipment Temperature:
Ambient |
| | f | | Species Identification Back to Top |
| 30330 | This procedure confirms the species of origin of the test article cells. Isoenzyme analysis is performed on extractions of clarified cell homogenates. For most cell lines, the procedure consists of comparing the electrophoretic mobilities on agarose gel films of test article and control enzyme extracts using four isoenzyme systems: glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and nucleoside phosphorylase (NP). Other isoenzyme systems are available for use when necessary. Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks which are filled almost to the top with medium, capped tightly and sealed with Parafilmâ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on the Sample Submission Form, as well as sufficient growth medium and supplements for two weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), media components and freezing conditions. Also list any drugs or special additives present in media.
· If cells are submitted cryopreserved, Protocol 30040 (Cell Growth for Assays) should be performed. | Cell Line Species Identity by Isoenzyme Electrophoresis
Turnaround Time:
3 weeks Container/Volume:
Minimum of 1 x 107 viable cells Shipment Temperature:
Ambient |
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| 30176 | This procedure confirms the species of origin of the test article cells. Isoenzyme analysis is performed on clarified cell homogenates. For most cell lines, the procedure consists of comparing the electrophoretic mobilities on agarose gel films of test article and control enzyme extracts using four isoenzyme systems: glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and nucleoside phosphorylase (NP). Analysis of human cell lines is similar, but involves the use of the following panel of isoenzymes to generate a phenotypic frequency profile or genetic signature: glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDH), phosphoglucomutase 1 (PGM1), phosphoglucomutase 3 (PGM3), esterase D (ESD), malic enzyme (ME-2), adenylate kinase (AK-1) and glyoxalase 1 (GLO-1).
The fluorescent antibody staining technique utilizes species-specific FITC-labeled antisera to confirm the identity of test article cells. A minimum of two antisera are employed for each test article. Usually 200 to 400 cells are examined with each antiserum. Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks which are filled almost to the top with medium, capped tightly and sealed with Parafilmâ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on the Sample Submission Form, as well as sufficient growth medium and supplements for two weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), media components and freezing conditions. Also list any drugs or special additives present in media.
· If cells are submitted cryopreserved, Protocol 30040 (Cell Growth for Assays) should be performed. | Cell Line Species Identity by Isoenzyme Electrophoresis and Immunofluorescence
(Reasearch test only)
Turnaround Time:
4 weeks Container/Volume:
1 x 107 viable cells Shipment Temperature:
Ambient |
| | f | | Master Cell Banks/Cryostorage Back to Top |
| 30033 | To prepare and preserve a Master Cell Bank (MCB), the cell line is subcultured, expanded, vialed and then frozen by a controlled rate process. The MCB is then analyzed for cell viability, growth, sterility, and the presence of mycoplasma. Notes
· If suspension cells, the appropriate number of viable cells should be sent in a tissue culture flask which is filled almost to the top with medium, capped tightly and sealed with Parafilmâ . Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures can be sent in a tissue culture flask as described above. Alternatively, frozen vials on dry ice can be shipped overnight.
· Growth requirements to expand and freeze cell are to be provided by the client.
· If preferred, client can provide raw materials directly for expanding and freezing cells. |
Creation of Master Cell Banks (GMP)
Turnaround Time:
10 weeks Container/Volume:
2 x 1 ml cryopreserved cells or
5 x 106 cells / ml Shipment Temperature:
Cryopreserved cells: On dry ice
Viable cells: Ambient |
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| 30764 | Cell line cryopreservation is a process used to assure an adequate supply of cells. A frozen supply of cells to draw upon preserves cells from changes due to genetic drift, cell line cross-contamination and adventitious agent contamination or from technical problems such as incubator failure. To prepare and preserve a GLP (or research) cell bank, the cell line is subcultured, expanded, vialed and then frozen by a cooling program that uses liquid nitrogen as the coolant. The cell bank is then analyzed for cell viability, growth, sterility (Protocol 30045) and the presence of mycoplasma using the Points to Consider test (Protocol 30055). For GMP Master Cell Banks, use Protocol 30033 (above). |
Cell Line Cryopreservation for GLP Cell Banks
Turnaround Time:
10 weeks
(Depends on growth of cells) Container/Volume:
2 x 1 ml cryopreserved cells or
5 x 106 cells / ml Shipment Temperature:
Cryopreserved cells: On dry ice
Viable cells: Ambient |
| | 33900 | Long-term storage of cell lines in vapor phase liquid nitrogen is provided for Master Cell Banks (MCB), Working Cell Banks (WCB), or critical cell lines. Cell lines are stored in validated, inventory-controlled liquid nitrogen storage units that are monitored 24 hours per day, 7 days per week to prevent storage unit or system failures. Notes
· Provide protocol for storage conditions and type of storage required (vapor/liquid). Also provide testing information on cell line for safety purposes (sterility, mycoplasma). |
Cryostorage of Biologic Specimens
Container/Volume:
1-2 ml cryovials
(Unique storage available) Shipment Temperature:
On dry ice |
| | f | | Cell Banking under GMP Guidelines Back to Top | Many aspects of the biopharmaceutical industry require mammalian cells and/or cell-based products as a component of their finished, final products. Continuing supplies of the specific cell lineage is integral to the manufacturing process. Cell banks are created to stock a supply of cells which have a comprehensive history that includes a documentation trail and safety testing as part of the overall validation process.
The GMP Cell Bank facility at AppTec offers clients an opportunity to have extensively characterized, professionally created cell banks grown to client specifications – under exacting standards and QA oversight – in a monitored, controlled environment. All work is SOP driven and documented. PROTOCOL DEVELOPMENT
With each cell bank, AppTec partners with the client to develop a client-specific, custom protocol. This is accomplished with direct, open communications between AppTec's scientific staff and the client’s tissue culture specialist. CELL EXPANSION AND HARVEST
Upon approval and sign-off on the protocol, the client submits the necessary sample to AppTec – usually one or two vials of cells. Cells are then expanded to the requisite density to fill the agreed-upon number of vials for the cell bank. The culture work is performed on a campaign basis in isolated ultra-clean tissue culture suites by fully gowned, highly skilled personnel. CRYOPRESERVATION, STORAGE AND SHIPMENT
Cells can be rate frozen to minimize freeze-related trauma.
Cells can be stored long term at AppTec in our GMP storage facility. Various levels of segregation can be reserved for your cell bank under vapor phase of liquid nitrogen.
Vials from the cell back can be shipped to the client as needed for manufacturing. Shipment of the entire bank should be in multiple individual transports. TESTING
Cell banks created at AppTec are tested for recovery viability, sterility and mycoplasma. Additional testing services are also available to completely characterize cell banks under FDA and ICH guidelines.
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