Tag Archives: top 10 biotech advances

Optimizing 2D Assay Kits for Use on 3D Cultures

Assay Optimization for 3D Cultures

No, not this 3D culture

3D Movie Culture

The culture of 3D movie goers.

But this kind

3D Cultures in a tissue culture context

Tissue culture cells growing in three dimensions.

Traditionally, mammalian cell culture means living cells grown outside the body on specially treated tissue culture plates in specialized incubators. Millions (dare I say billions?) of experiments utilizing this technique leading to huge advances in research and medicine.

To improve on this convention, innovators develop cultures that grow, not just in two dimensions (2D), but three dimensions (3D). General consensus in the field now is that these 3D cultures are more physiologically relevant—closer to native whole organisms—than conventional 2D cultures.

With more use of 3D cultures in business and research, a new challenge to testing larger volumes of cells arises. Almost all previous assays used to test qualities of cells in culture have only been tested and optimized for traditional 2D models. What hurdles face scientist who want to test (assay) their cultures in 3D rather than 2D?

Terry Riss, a Promega scientist, presented his company’s findings in a talk at the Society of Toxicologist meeting on Monday, March 23, 2015 at the San Diego Convention Center. Promega’s work has been primarily conducted with spheroids generated with a hanging drop method from a company aptly named In Sphero

In thinking about differences between 2D and 3D cultures, one huge differences is the ability of reagents to diffuse longer distances into cells. Two dimensional cells tend to grow flat and spread out on the dish surface, allowing great accessibility to the innards of the cells, which scientist are obsessively interested in.

Promega offers various “Glo” assays for cell viability. Generally they are better than the usual MTT or resazurin tests in that they are less toxic to the cells and permit the same cells to be used again after the assays for even more assays (there we go again, us scientist and our obsession with assays).

In general, Riss advocates optimizing any off-the-shelf assays developed for 2D cell culture with your own particular cell line and application (basic good lab practice in my book!).

Try increasing these three parameters:

  • Detergent concentration to lysis the cells
  • Physical disruption used to dissociate the cells
  • Time of incubation

As always, remember to  include controls, both positive and negative, and optimize the experiment to your particular assay needs.

As the drug development moves towards more 3D cell culture models, the need for assays of these cultures will grow.  Promega is adding to their repertoire of kits to meet this need.

San Diego Clinical Research Network

Did you miss the San Diego Clinical Research Network (SDCRN) meeting Monday June 24 on the top 10 innovations in biotech?  Keep reading for a summary.

Karin Lucas, PhD, was the speaker for the evening.  She is the Director of Education & Training for BioTechPrimer developing curriculum geared towards the non-scientist (HR, legal, and business) interested in the biotech field. Biotech Primer offers training onsite or offsite, customizable training, and online formats.  Founded in 2001, BioTechPrimer offers classes internationally, having provided instruction in China, Scotland, Germany, France, and Australia.

Karin began with a quick review of the central dogma of biology which states that information flows from DNA to RNA to protein.  Then she began the meat of her talk:

 Top 10 Biotech Advances

1)  Regulatory function of intergenic DNA

Initially thought to be “junk DNA”, because scientists did not understand its function, DNA between genes is now described as intergenic DNA.  Comprising over 95% of the human genome, intergenic DNA is involved in gene regulation, determining when and where a specific protein is made.  SNPs (single nucleotide polymorphism—pronounced “snips”) are often used as markers for diseases; 90% of SNPs that correlate to disease are located in the intergenic region.  Hence, this “junk DNA” fulfills a specific and important task:  regulating protein expression.

2)  Speed and cost of DNA sequencing:  biotech beats Moore’s law

Moore’s Law states that the number of transistors on an integrated circuit doubles approximately every two years.  The transistor number is closely tied to an increase in computing speed, memory available, even pixels in a digital camera.

Back in the dark ages (circa 1990), DNA sequencing in a research lab was considered efficient if one scientist deduced 300 base pairs of DNA in one day.  Compare this to the standards of today, when the entirety of a genome (3 BILLION base pairs) is sequenced in one week!

genome sequencing costs

While the efficiency of microprocessors increases exponentially with time, the rate of decrease in the cost of DNA sequencing is even greater.

Looking at the cost of DNA sequencing, the first human genome, published in 2003, cost $3 billion.  In 2007, the cost for one human genome was reduced to $1 million dollars; today the cost is below $7000.

The ease with which DNA can be sequenced has also been enhanced.  The days of huge glass plates, radioactive nucleotides, and neurotoxic gels are ancient history.  Present day DNA sequencers take up a small foot print and can easily be run by an individual with an associate degree.

However, we are still a long way from being able to hand a typical family doctor a thumb drive with your genomic sequence on it and having them have any idea what to do with it.  Several companies in the San Diego area are working hard to bring practical applications of whole genome sequencing to the bedside.

3)  Stratified Medicine

The concept here is to tailor medicine to each individual patient with companion diagnostics.  Drug companies are working to determine how a treatment affects a person individually based on biomarkers—unique characteristics found in a person’s DNA, RNA and/or proteins.  Thus, it will be known in advance how a person will respond to a particular drug; more effective pharmaceutical can be used first instead of randomly guessing which will be the most effective treatment.

4)  Human Microbiome

bacteria in human gut

Bacteria in the human gut aid digestion.

I’m really covered by bacteria?  Yes!  For every human cell in our bodies, we have 10 non-human cells in the form of bacteria, yeast and molds.  Microbes have an effect on chronic inflammation, atherosclerosis, cancer, and diabetes.  Maybe in the future, we will only have to adjust our microbiome to manage these conditions.

5) Controlling Protein Expression by inhibiting RNA

Often a disease state is induced by the expression of the wrong protein, during the wrong time, in the wrong place, or in the wrong quantity.  This can be remedied by getting rid of RNA for a particular protein.  Once this is accomplished with double-stranded RNA, the mechanism continues to destroy those faulty proteins.  A local company, ISIS recently received FDA approval to use RNA interference to treat familial hypercholesterolemia.

6)  Gene Therapy

Gene therapy

Note: picture not to scale

Gene therapy was first used in 1990 to treat severe combined immunodeficiency disorder (SCID).  In 2000 retroviruses were used for SCID; of the 20 persons receiving treatment, about half were cured and while the other half got leukemia.  Hence, the FDA has been reluctant to approve further human trials, much less treatments.  In 2012, however, the EMA (European equivalent of the FDA) approved Glybera from UniQure to treat the rare genetic disorder lipoprotein lipase deficiency.  This treatment specifically infects only muscle cells and must be repeated every few years.  Still it is good progress for a treatment long in development.

7)  Stem cell treatments

These treatments offer hope for neurodegenerative diseases like ALS. Other companies are in clinical trials for spinal cord injuries and stroke.  These stem cells can arise from either embryonic stem cells or from adult stem cells.

8)  Human cloning advance

cloning results may vary

Cloning: Results may vary

While many researchers have thrown their hands up in despair at ever succeeding in cloning a human, workers at the Oregon Health and Science University persevered.  With patient tinkering they successfully created cloned blastocytes.  By taking a part of the developing zygote, stem cell lines were created.   These could then be differentiated into a variety of different cell types.  One day, this breakthrough could have great therapeutic potential since the genes from these cells match perfectly with this donor of the genetic material eliminating the risk for rejection of replacement tissues.

9)  Bionic eye

bionic eye

It is possible for the blind to see again.

Argus II device by Second Site bypasses damaged photoreceptors in the eye, giving legally blind persons the ability to recognize faces and read four letter words.  This device won FDA approval in 2013; it has been available in Europe since 2013.

10) Biofuels

green crude farm

Sapphire Energy green crude farm.

Corn and soy, the sources of conventional biofuels, require good soil to grow.  Algae on the other hand, can grow in salt water.  To merely replace diesel fuel in the US with biodiesel would require all the arable land in the US to be cultivated with soy.  By occupying a site only the size of Georgia, algae could replace all the biodiesel in the US.

The last hurdle to biofuels is the price.  Sapphire Energy is targeting $75 per barrel for the green crude produced in their New Mexico plant, due to open in 2014.  This product has the added benefit of being able to be processed as light sweet crude oil in un-modified refineries alongside with regular crude oil.

More details on the meeting…

Teresa Gallagher founded the SDCRN, in response to a need for a group looking at clinical research programming.  Several other groups in San Diego already address the needs of entrepreneurs, businesses, and biotech.  Her vision is to bring together groups of persons working in several different areas from contract research organizations (CROs), to academic institutions, to other companies in the greater San Diego area.  Membership in San Diego Clinical Research Network is open to individuals working in the life science industry or healthcare in San Diego and Orange Counties.  Since starting SDCRN on LinkedIn, her group has grown to more than 500 members.  Teresa encourages feedback on what sort of programming the community would like to see at SDCRN events relating to clinical development.  What issues in clinical development arise in your work and what aspects of them would you like help with?

The Del Mar Offices of Sheppard Mullin law firm hosted the evening. The venue was fantastic and the food even better.  Shepherd Mullin is a worldwide law firm with practice areas in intellectual property, corporate, food and drug regulatory, and more.  Additionally, Shepherd Mullin publishes a FDA law blog with information on current issues affecting FDA-regulated companies.

DeeAnn Visk, Ph.D., is a freelance science writer, editor, and blogger. Her passions include cell culture, molecular biology, genetics, and microscopy. DeeAnn lives in the San Diego, California area with her husband, two kids, and two spoiled hens. You are welcome to contact her at deeann.v@cox.net