VE-BEVS Cell Line Resource Page

Instructions for Product No.: 10010; 10020; 10030

Content

Product No. 10010, 10020 and 10030 contain >1×10⁷ cells in 50% fresh Sf-900 II serum free medium (SFM; GibcoTM), 50% conditioned Sf-900 II SFM and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5%.

Shipping and Storage

Cells are shipped on dry ice and are supplied in a cryogenic vial containing >1x10⁷ cells/mL. Cells were frozen in a freezing medium composed of 50% fresh Sf-900 II SFM, 50% conditioned Sf-900 II SFM and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5%. Store cells in liquid nitrogen (vapor phase).

Live cells can be shipped upon request.

Caution: DMSO is a hazardous material and caution has to be taken when handling this substance.

Product Qualification

To qualify for sales cells must be in logarithmic growth with 98% viability and less than 20 passages before they are frozen. Cells have been shown to recover as healthy logarithmically growing cells within 3 days after thawing.

Cell Maintenance and Handling of Cells

Medium Requirement:
Use of Sf-900 II SFM (Gibco) is recommended but cells also grow well in TNM-FH Insect Cell Culture Medium supplemented with 10% heat-inactivated FBS. NOTE: The addition of 400µg/mL G418 is not essential and might interfere with trypan blue staining of cells. WE RECOMMEND STARTING THE CELLS IN ADHERENT CULTURE AND THEN ADAPTING TO SHAKER CULTURE ONCE ESTABLISHED. IF THE CELLS DO NOT APPEAR TO BE VIABLE, DO NOT ATTEMPT TO REVIVE THE SECOND VIAL OF CELLS PROVIDED UNTIL YOU HAVE CONTACTED PARATECHS CORPORATION FOR ADVICE.

Thawing Cells:
Thaw frozen cells rapidly. Decontaminate the outside of the vial with 70% ethanol before transferring the 1 mL cell suspension into one T-25 cm2 flask.

Adherent Culture:
Add 1 ml of Sf-900 II SFM into the cryovial containing thawed cells. Gently resuspend the cells. Remove the cells from the cryovial and add to a T25 flask containing 4 mL of medium. Transfer flask to a 27°C incubator and allow the cells to attach for 45-60 minutes before replacing the medium with 5 mL fresh Sf-900 II SFM. Subculture cells when they have reached a density of >80% confluency. Release cells from the flask’s surface by tapping the flask sharply against your palm until >75% of the cells have detached and transfer 2 mL cells into each of two new T25 flask containing 3 mL of medium. When these flasks have reached a density of >80% confluency, pass as above to generate a total of four flasks of cells. Let grow to >80% confluency and proceed to Suspension Culture.

Suspension Culture:
To start a suspension culture, release the cells from three T25 monolayer cultures and transfer the entire volume (a total volume of 15 mL) into a 125 mL shaker flask containing 15 mL of fresh Sf-900 II SFM. It is recommended that the cell line also be continued as adherent culture in a T25 flask as a back-up source of cells.
Incubate shaker flask in a 27°C incubator on an orbital shaker platform rotating at 100-110 rpm. Loosen caps of flasks to allow proper oxygenation/aeration. Allow the cells to grow for 3-4 days. Count the cells from the starter flask and transfer the volume of cells necessary to reach a seeding density of 1×10⁶ cells/ml in 50 mL of Sf-900 II SFM in a 125 mL shaker flask. Once a suspension culture has been established and a cell density of 5-8×10⁶ viable cells/mL has been reached VE cells are routinely diluted to a cell density of 0.8-1×10⁶ viable cells/mL with Sf-900 II SFM.

VE cells have an average diameter of approximately 21 µm which is bigger than Sf9 cells. In addition, VE cells grow slower than Sf9 cells.

• VE-CL-01 Insect Cells (Product No. 10010) have a cell doubling time of 37.6 hrs in Sf-900 II SFM and an average cell size of 21.6 µm.

• VE-CL-02 Insect Cells (Product No. 10020) have a cell doubling time of 29.7 hrs in Sf-900 II SFM and an average cell size of 21.3 µm.

• VE-CL-03 Insect Cells (Product No. 10030) have a cell doubling time of 43.6 hrs in Sf-900 II SFM and an average cell size of 21.7 µm.

Freezing Cells:

Freeze cells at a density of >2×10⁷ viable cells/mL in a freezing medium composed of fresh Sf-900 II SFM, 10% heat-inactivated FBS and DMSO to a final concentration of 7.5%. (Optional freezing media: 50% conditioned Sf-900 II SFM: 50% fresh Sf-900 II and DMSO to a final concentration of 7.5%). Centrifuge cells at 100g at 4°C for 5-10 minutes, remove the supernatant and resuspend the pellet in an appropriate volume of chilled freezing medium to reach a density of >2×10⁷ viable cells/mL. Transfer suspension into a cryovial.  Place cells in a styrofoam container and place at -20°C for one hour, then transfer the styrofoam container with cells to –80°C overnight before transferring the cells to liquid nitrogen (vapor phase). Frozen cells remain viable if properly stored in liquid nitrogen.

Overview of Vankyrin-Enhanced (VE) Insect Cell Line

Vankyrin-Enhanced Insect Cells (VE cells) are transgenic insect Sf9 cells that have been engineered to stably express the Campoletis sonorensis ichnovirus P-vank-1 protein (Fath-Goodin et al., 2006; Kroemer and Webb, 2006; see below). Sf9 cells originated from the IPLBSF-21 cell line, derived from the pupal ovarian tissue of the fall army worm, Spodoptera frugiperda [Vaughn et al., 1977; Vaughn, J.L., Goodwin, R.H., Tompkins, G.J. and McCawley, P. (1977). The establishment of two cell lines from the insect Spodoptera fugiperda (Lepidoptera: Noctuidae)].

The stably transformed VE insect cell line was obtained by transfecting Sf9 cells with ParaTechs’ proprietary transformation vector harboring the P-vank-1 gene and the neomycin resistance gene. Geneticin G418 Sulfate was then used to select for stable cell lines. The expression of the P-vank-1 transcript was confirmed by RT-PCR.

The presence of the P-vank-1 protein leads to prolonged longevity and increased recombinant protein production of baculovirus infected VE cells compared to regular Sf9 cells.  This cell line has been developed for enhanced recombinant protein production using the baculovirus expression vector system (BEVS).

• Modified insect Sf9 cells stably expressing a Campoletis sonorensis ichnovirus vankyrin gene

• Use of G418 for selection of stable lines

• Prolonged longevity of cells after infection with a BEVS

• Up to 14-fold increase in protein yield as compared to regular Sf9 cells. Further enhancement (up to more than 20-fold) can be obtained by using modified cells in combination with the VE-BEVS transfer vector.

• Compatible with all conventional BEVS.

• Essentially no additional work or adaptation required.

• Expression of recombinant protein may need to be optimized by testing different time points and MOIs.

This product is intended for research purposes only.

CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

ParaTechs Corporation Limited Warranty

ParaTechs warrants that, at the time of shipment, the Product will conform to the specifications that accompany the Product.  This warranty limits ParaTechs liability to replacement of the Product.  PARATECHS MAKES NO OTHER WARRANTIES, EXPRESSED OR IMPLIED, WITH RESPECT TO THE PRODUCT; INCLUDING ANY WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE OR THAT THE PRODUCT DOES NOT INFRINGE ANY PROPRIETARY RIGHTS OF ANY THIRD PARTY.

1. What cell line were the VE-cell lines derived from?

The VE-cell lines are engineered from Sf9 cells that were originally derived from the ovarian tissue of the fall armyworm (Spodoptera frugiperda).

2. Do I need to keep the VE-cell lines under antibiotic selection to prevent loss of expression of the vankyrin protein?

No, the VE-cell lines are stably transformed cell lines and antibiotic treatment is not needed to maintain vankyrin protein expression.

3. What is the average size of the VE-cells?

While derived from Sf9 cells the VE-cells are larger in size than the parental Sf9 cells. VE-cells are approximately 21 microns in diameter.

4. What media are VE-cells frozen in?

Cells were frozen in 50% fresh Sf-900 II serum free medium, 50% conditioned Sf-900 II serum free medium and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5%

5. How many passages may I maintain my cells?

We recommend starting new cells after they have been through 30 passages. A passage is considered anytime you add media to dilute your cells.

6. How do I start my VE-cell lines?

Thawing Cells:

Thaw frozen cells rapidly in a 37°C water bath. Decontaminate the outside of the vial with 70% ethanol before transferring the cells into one T-25 cm² flask. We recommend starting the cells in adherent culture and then adapting to shaker culture after 2 passages.

Adherent Culture: Add 1 mL of Sf-900 II serum-free medium (SFM) into the cryovial containing thawed cells. Gently resuspend the cells. Remove the cells from the cryovial and transfer into one T25 flask containing 5 mL of medium. Transfer flasks to a 27°C incubator and allow the cells to attach for 45-60 minutes before replacing the medium with 5 mL fresh Sf-900 II SFM. Subculture cells when they have reached a density of >80% confluency. Release cells from the flask’s surface by tapping the flask sharply against your palm until > 75% of the cells have detached and transfer 2 mL cells into a new T25 flask containing 3 mL of medium.

Suspension Culture: To start a suspension culture, release the cells from two T25 monolayer cultures and transfer the entire volume from one flask and 3 mL from the second flask (a total volume of 8 mL) into a 125 mL shaker flask containing 12 mL of fresh Sf-900 II SFM. Use the remaining 2 mL of cells to continue the cell line as adherent culture in a T-25 flask.

Incubate Erlenmeyer flask in a 27ºC incubator on an orbital shaker platform rotating at 100-110 rpm. Loosen caps of flasks to allow proper oxygenation/aeration. Allow the cells to grow for 3-4 days. Count the cells from the starter flask and transfer the volume of cells necessary to reach a seeding density of 1×10⁶ cells/mL in 50 mL of Sf-900 II SFM in a 125 mL shaker flask. Once a suspension culture has been established and a cell density of 5-8×10⁶ viable cells/mL has been reached VE cells are routinely diluted to a cell density of 0.8-1×10⁶ viable cells/mL with Sf-900 II SFM.

7. How do I freeze my cells down?

Freeze cells at a density of >2×10⁷ viable cells/mL in a freezing medium composed of 50% fresh Sf-900 II serum free medium (SFM), 50% conditioned Sf-900 II serum free medium and Dimethyl Sulfoxide (DMSO) to a final concentration of 7.5% [optional freezing media: fresh Sf-900 II SFM (Invitrogen™), 10% heat-inactivated FBS and DMSO to a final concentration of 7.5%]. Centrifuge cells at 100g at 4°C for 5-10 minutes, remove the supernatant and re-suspend the pellet in an appropriate volume of chilled freezing medium to reach a density of >2×10⁷ viable cells/mL. Transfer suspension into a cryovial. Place cells in a styrofoam container and place at -20°C for one hour, then transfer the styrofoam container with cells to -80°C overnight before transferring the cells to liquid nitrogen (vapor phase). Frozen cells remain viable if properly stored in liquid nitrogen.

8. Which VE-cell line should I purchase?

VE-CL-01 Insect Cell Line (Product #10010) displays the most prolonged enhanced protein expression (up to 7 days post-infection) when using conventional BEVS viruses. We recommend VE-CL-01 for laboratories working with highly stable intracellular and secreted proteins as the pronounced enhancement of viability in these cells allows for prolonged expression and accumulation of recombinant protein over time.

VE-CL-02 Insect Cell Line (Product # 10020) displays a sharp peak of enhanced protein expression at day 3 and day 4 post-infection. We recommend VE-CL-02 for laboratories working with highly unstable or toxic proteins as enhanced protein expression occurs at a very high level and over a very short time interval in these cells.

VE-CL-03 Insect Cell Line (Product # 10030) displays an intermediate phenotype to VE-CL-01 and VE-CL-02 showing significantly enhanced protein expression and moderately enhanced longevity. Due to its intermediate expression properties, we recommend VE-CL-03 for laboratories working with proteins of unknown toxicity and stability or for general enhancement of most recombinant proteins expressed in conventional recombinant BEVS viruses.

Steele KH, Stone BJ, Franklin KM, Fath-Goodin A, Zhang X, Jiang H, Webb BA, Geisler C (2017). Improving the baculovirus expression vector system with vankyrin-enhanced technology. Biotechnol Prog. 2017 Jun 26. doi: 10.1002/btpr.2516. PMID: 28649776. https://www.ncbi.nlm.nih.gov/pubmed/28649776

Lee SM , Wu CK, Plieskatt J, McAdams DH, Miura K, Ockenhouse C, and King CR. (2016). Assessment of Pfs25 expressed from multiple soluble expression platforms for use as transmission‑blocking vaccine candidates. Lee et al. Malar J (2016) 15:405, DOI 10.1186/s12936-016-1464-6. https://malariajournal.biomedcentral.com/articles/10.1186/s12936-016-1464-6

Fath-Goodin A, Kroemer JA, Webb BA (2009). The Campoletis sonorensis ichnovirus vankyrin protein P-vank-1 inhibits apoptosis in insect Sf9 cells. Insect Mol Biol. 18(4):497-506. PMID: 19453763. https://pubmed.ncbi.nlm.nih.gov/19453763/

Fath-Goodin A, Kroemer J, Martin S, Reeves K, Webb BA (2006). Polydnavirus genes that enhance the baculovirus expression vector system. Adv Virus Res. 68:75-90. Review. PMID: 16997009. https://pubmed.ncbi.nlm.nih.gov/16997009/

Kroemer JA, Webb BA (2006). Divergences in protein activity and cellular localization within the Campoletis sonorensis Ichnovirus Vankyrin family. J Virol. 80(24): 12219-28. PMID: 1700565. https://pubmed.ncbi.nlm.nih.gov/17005654/

Steele K, oral presentation entitled “Expression of mammalian glycoproteins and other difficult to express proteins with the vankyrin-enhanced baculovirus expression system.” Presented during PEGS: The Essential Protein Engineering Summit, Boston, MA, May 6, 2014 [PDF]

Steele K, poster presentation entitled “End the poor protein yield crisis by delaying cell lysis: new baculovirus transfer vectors encoding the anti-apoptotic vankyrin protein.” Presented during the International Society for BioProcess Technology, 4th Spring meeting, Washington D.C., March 12, 2014

Oral presentation entitled “Function and Versatility of Vankyrin Gene-Mediated Enhancement of Baculovirus Expression Vector Protein Production” at ISBiotech 2nd Annual Meeting “Baculovirus Expression Technology”, Rosslyn, VA, April 2-5, 2012

Poster presentation entitled “Log-Scale Improvement in Protein Yield with Vankyrin-Enhanced Transfer Vector” at the Cambridge Healthtech Institute “Baculovirus Technology” conference, Boston, MA, August 22-23, 2011 [PDF]

Oral presentation entitled “Expanding the Utility of Viral Ankyrin Genes in Baculoviruses” at the Wilbio 12th International Conference on Baculovirus and Insect Cell Culture, San Antonia, TX, February 2-4, 2009

Oral presentation entitled “Improved Recombinant Protein Production by ParaTechs’ Vankyrin-Enhanced Baculovirus Expression Technology” ” at the Cambridge Healthtech Institute “Baculovirus Technology” conference, Boston, MA, August 2008

Poster presentation entitled “Enhanced Recombinant Protein Production by ParaTechs’ Vankyrin Enhanced Baculovirus Expression Technology” at the Wilbio 11th International Conference on Baculovirus and Insect Cell Culture, Seattle, WA, February 25-27, 2008 [PDF]

The VE-BEVS Cell Line (“Product”) and VE-BEVS Transfer Vectors (“Product”) were developed in collaboration by scientists at ParaTechs and the University of Kentucky Lexington for expression of recombinant proteins.  One or more patents or patent applications owned by the University of Kentucky Lexington cover components of the Product.

ParaTechs Corporation has an exclusive license to sell the Product to scientists for academic research or one year commercial evaluation only, under the terms described below. Use of the Product for any Commercial Purpose (as defined below) other than evaluation requires the user to obtain a commercial license as detailed below. Before using the Product, please read the terms and conditions set forth below. Your use of the Product shall constitute acknowledgement and acceptance of these terms and conditions. If you do not wish to use the Product pursuant to these terms and conditions, please contact ParaTechs Technical Service Department to return the unused and unopened Product for full credit.

ParaTechs grants the purchaser a non-exclusive license to use the enclosed Product for academic research or for commercial evaluation purposes only. The Product is being transferred to you in furtherance of, and reliance on, such license. You may not use the Product, or the materials contained therein, for any Commercial Purpose without a license for such purpose from ParaTechs Corporation.

Commercial Purpose include any use of Product in a Commercial Product, the manufacture of a Commercial Product, any resale of the Product, any use (other than evaluation) of Product to facilitate or advance research or development of a Commercial Product, and any use (other than evaluation) of the Product to facilitate or advance any research or development program the results of which will be applied to the development of Commercial Products.

 “Commercial Product” means any product intended for commercial use.

 Access to the Product must be limited solely to those officers, employees and students of your entity who need access to perform the aforementioned research or evaluation. Each such officer, employee and student must be informed of these terms and conditions and agree in writing, to be bound by same. You may not distribute the Product to others. You may not transfer modified, altered, or original material from the Product to a third party without written notification to and written approval from ParaTechs.

Inquiries for commercial use should be directed to agoodin@paratechs.com.

Patent information:
VE-BEVS Cell Line United States Patent 7,842,493 [PDF]
VE-BEVS Transfer Vector United States Patent 7,629,160 [PDF]