Stable Cell Line Development Service

Stable Cell Line Development Service

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Research Lab
Research Lab
Fermentation Room
Fermentation Room
Purification Room
Purification Room
Large-Scale Cell Culture Room
Large-Scale Cell Culture Room


SPEED BioSystems Research
and Production Facility

Stable Cell Line Development services for research and biopharmaceutical production

Our state-of-the-art technologies and world-class expertise transform to significantly reduced time, effort and costs in developing high-performance mammalian cell lines for production of recombinant proteins and antibodies.

Host cells: CHO, HEK293, Hela or customer designated.

Genes-of-Interest: Antibodies, GPCRs, Ion channels, Proteases, Kinases, Trans-membrane proteins, Adhesion molecules

Full Service: Choosing part or all of the following services based on your specific needs

  • Cloning your gene-of-interest into our proprietary high-expression vector (5-10 times more potent than traditional mammalian vectors)
  • Transfection or multiple transfections
  • Drug Selection and Gene Amplification (Antibiotics, DHFR, GS, etc.)
  • Screening of high-expression clones
  • Clone expansion and banking
  • Stability test
  • Medium optimization
  • Fermentation process development
  • End product production for animal tests or clinical trials

Research and Development enabling and Project management: Protein drugs and antibodies

If you are a newly started company without your own lab set up yet, or you are a big company looking to improve your R&D capabilities, we can take care of most of your pre-clinical researches: from gene cloning, cell-line development, protein/antibody purification, all the way to product manufacturing. Just give us the sequence of your gene-of-interest, sit back have a cup of coffee and you will have your final product ready in no time. We offer the following services:

  • Molecular cloning of the gene of interest into our proprietary expression vector
  • For antibody: CDR grafting, Humanization, Isotype switching, Affinity Maturation, etc
  • Transfection and selection of stable cell lines
  • Gene amplification to make high expression clones
  • Adaptation to serum- and protein-free medium
  • Cell banking
  • Clonal stability testing
  • Fermentation process development
  • Purification process development
  • Manufacturing
  • In vitro research

Please contact us at cell-line@SpeedBioSystems.com for more information and a quote.

For a quotation, please fill in the form and send it back to cell-line@SpeedBioSystems.com.

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Stable Cell Line Development Learning Center

Contents


Why develop stable cell lines
How to develop stable cell lines
How does SPEED BioSystems develop stable cell lines
Frequently asked questions
A general protocol for making stable cell lines

Why develop stable cell lines

Stable cell lines are immortal cells that can “stably” express a gene in a controlled level, either more than, or less than, the gene’s physiological normal levels. The cell lines should be able to grow continuously over a prolonged period of time without considerable change of target gene’s expression levels.

Stable cell lines are widely used in functional studies of genes, drug screening and production of proteins, for both therapeutic and non-therapeutic purposes.

  • Functional studies: the expression level of a gene in certain cells can be manipulated to study its functions. A strong promoter can be used to over-express the gene in a stable cell line. Similarly, shRNAs, anti-sense RNAs and ribozymes can also be used to knockdown the gene in stable cell lines. Those stable cell lines are used every day in many gene function research labs across the world. Because the above-mentioned stable cell lines are mainly used for research purposes, the quality requirements are low. Usually there is no need to test stability or virus contaminations in those stable cell lines.
  • Drug screening: Stable cell lines are very important in screening drug candidates out of a combinatorial chemical library. Such screenings are performed automatically by mechanical robots. The readout of a positive hit is usually a change of some light signals due to protein-drug interactions. Stable cell lines expressing target genes and some light-sensitive proteins (such as GFP) are essential to such screening tasks. Those stable cell lines are usually very complicated. They might express 2-10 different genes in a single cell.
  • Production of Research proteins: Proteins for research purposes can be produced by transient transfection of HEK293 cells, which can be efficiently transfected with many methods. Many of SPEED BioSystems’ catalog proteins are produced this way. However, this method is not very cost effective in producing recombinant proteins in large quantity. For some highly demanded proteins, a stable cell line is more desirable as production host. For example, large amount of human cytokines are used frequently in biomedical researches. To meet the high demand of high quality cytokines produced by human cells, SPEED BioSystems developed several stable HEK293 cell lines for production of those cytokines. For a list of available human cytokines produced by stable HEK293 cells, please click here.
  • Production of Pharmaceutical proteins/antibodies: Stable cell lines are most useful for making recombinant proteins, recombinant antibodies and vaccines for therapeutic or preventive purposes. Because those products are going to be used in humans, how they are produced is strictly regulated by the FDA. In most cases, the products cannot pass FDA approval unless they are produced by stable cell lines. Creating a stable cell line for production of therapeutics is usually a long process that requires specialized skills and expertise that can only be acquired from previous experience. Although many cells can express recombinant proteins and antibodies efficiently, CHO (Chinese hamster ovary) cells are most frequently used as the host for producing FDA-regulated therapeutics. A lot of fermenter configurations, cell culture medium recipes, and downstream purification methods are designed around using CHO as production host in large-scale cell culture. Not surprisingly, many of SPEED BioSystems’ contract stable cell lines are also based on CHO cells.
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How to develop stable cell lines

To make an over-expression stable cell line, the gene-of-interest is usually put under the control of a strong promoter and then transfected into a host cell line. The target gene can stay out of host chromosomes and replicate independently as “episomes”. However, such a cell line is not very stable. The gene-of-interest could be lost over a period of continuous culture due to cell divisions, even under antibiotic pressure. To make a truly stable cell line, the gene-of-interest has to be integrated into host chromosomes, where the gene-of-interest can be multiplied and then divided into daughter cells together with host genetic materials. In such stable cell lines, the integrated genes can remain intact over very long period of continuous culture, even without any antibiotic pressures.

Target genes can be inserted into host cells’ chromosomes through:

  • Random integration: A plasmid containing the gene-of-interest and selection markers is transfected into host cells. The plasmid will integrate into host genome at random locations. Such integrations are very rare events. To make sure that a stable cell line with desirable expression level and stability can be obtained, large number of cells must be selected and screened following transfection. Sometimes the exact integration location also need to be determined to make sure that no important host genes are disrupted because some genes are essential for maintaining cell viability when the cells are cultured in large fermenters for subsequent protein production. The randomly integrated genes can be amplified using dihydrofolate reductase (DHFR) system or glutamine synthetase (GS) system. After amplification, gene copy numbers per cell can increase as much as 1000-fold. Almost all CHO stable cell lines used in biopharmaceutical production are developed with random integration followed by several rounds of gene amplification.
  • Targeted integration: In contrast to random integration where the exact integration locations are uncontrollable, targeted integration allows precise integration into pre-determined locations. Such integrations are usually mediated by DNA recombinases, such as Cre-Lox system and Flp-FRT system. For example, Invitrogen’s “Flp-In™-CHO Cell Line” is based on Flp-FRT system. Making a stable cell line with targeted integration is much faster than random integration. However, stable cell lines made with targeted integration have single copy of target gene per cell and the integrated genes cannot be amplified, limiting the expression level of foreign proteins and making such cell lines unsuitable for production of therapeutics.
  • Virus-induced integration: Some viruses such as adenovirus, retrovirus and lentivirus are known to be able to integrate their genomes into host cells. When a foreign gene is inserted into the genome of one of those viruses, the foreign gene can be integrated into the chromosomes of host cells along with the virus genome, thus creating a stable cell line harboring the foreign gene. Stable cell lines made this way are usually used for research only. They are not suitable for production of therapeutic proteins due to safety issues.
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How does SPEED BioSystems develop stable cell lines

At SPEED BioSystems, we make all kinds of cell lines using various methodologies as summarized above. Contrary to many of our competitors’ claims, we do not possess any “magic tricks". We make stable cell lines using classical protocols that have been proven most effective by ourselves and by our fellow scientists. The mission of SPEED BioSystems’ stable cell line development service is relieving our customers from tedious, time-consuming works so they can focus on other important projects. We do not intent to replace our customers as the designer of their projects, although we do offer suggestions to them based on our experience. Before we send out a quote, we usually talk with our customers over phone to understand their goals and then offer the best suggestions on how to achieve the goals while at the same time save their precious time and money.

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Frequently asked questions

Q: How much does it cost to generate a stable cell line?
A: Well, it depends. It is determined by many factors, such as target genes, host cell lines, expression levels and how soon do you need to have the stable cell line, etc. Usually we discuss with our customers over phone before we send out a detailed quote.

Q: How long does it take to create a stable cell line?
A: We deliver most stable cell lines to our customers within 4 - 6 weeks. Again, it is determined by many factors. We can give you a more precise estimate after a conversation over phone.

Q: How many clones do you screen for a positive stable clone?
A: We usually screen 10 96-well plates for positive clones. We may screen more clones based on specific needs of individual projects.

Q: Does SPEED BioSystems guarantee success?
A: We have > 90% success rate in making stable cell lines. However, in some rare occasions, a stable cell line cannot be generated due to gene toxicity, gene-host incompatibility and many other reasons. In that case, we share risks with our customers by waiving 50% of quoted price.

Q: What is the payment schedule for your stable cell line service?
A: We will need to receive prepayment of half of the quoted price before we can start working on the project. This charge will be used to cover part of materials cost and labor cost, and is not refundable. The other half of quoted price is due immediately after the generated cell lines are shipped.

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A general protocol for making stable cell lines

If you want to develop stable cell lines on your own, below is a general outline of how to generate stable cell lines:

  1. Vector construction: Clone your gene-of-interest into a mammalian cell expression vector. The vector should contain a strong promoter (such as a CMV promoter) and at least one selection marker. The selection marker can be a drug resistant gene (such as genes resistant to G418, Puromycin or Hygromycin, etc) or a fluorescent protein (GFP, YFP, RFP, etc).
  2. Transfection: Purify the expression vector and transfect it into your target host cells. You should select the transfection method that is most suitable for your target cells. For CHO, HEK293 and Hela cells, Lipofectamine™ and similar transfection reagents usually give satisfactory results. For some hard-to-transfect cells, you might try electroporation, such as Lonza’s Nucleofector™ Technology. There is always a compromise between high transfection efficiency and cell viability rate.
  3. Selection: If the selection marker is a fluorescent protein, then you can sort out your target cells with FACS (fluorescence-activated cell sorting). FACS machine is expensive and is not available in most labs. Probably it is easier to select your target stable cell lines with antibiotics. In that case, you can grow your cells in medium containing a range of antibiotics (such as G418) concentrations (e.g. 250 μg/ml, 500 μg/ml, 1 mg/ml and etc) until 99% of the cells are dead. Then collect the leftover live cells, count them, and plate the cells into at least ten 96-well plates. Make sure each well contains only one cell. Culture the cells continuously with medium containing antibiotics for 7-10 days until colonies begin to appear.
  4. Screening: You can use many methods to assay the selected cell lines that express highest level of your target proteins. For secreted proteins and antibodies, you may use ELISA to screen out the best performing stable cell lines. For membrane proteins, you may use flow cytometry or cell surface ELISA. For intracellular proteins, you may have to use Western Blot. The best screening method must be determined by the researchers.
  5. Expansion and freezing down: Once the highest expression clones are identified, you will need to expand the clones, and freeze them down for later use. If the stable cell lines are going to be used for producing therapeutics, additional assays will need to be performed in a GMP facility, such as stability, virus and mycoplasma contamination, large-scale cell culture process development, etc.
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