BioTether Sciences has a Lonza 4D-Nucleofector with X unit. This device enables users to efficiently transfect immortalized cells, primary cells, stem cells and other hard to transfect cell lines with high efficiency. Experiments are designed to use the standard cuvette or a 16 strip microcuvette. The X unit can electroporate 2 standard cuvettes at the same time, and both are able to be programmed independently from each other. The 16 strip microcuvette is great for optimization, and each microcuvette can be programmed independently as well. This allows for optimization of electroporation conditions to identify the highest efficiency  (up to 90%) and lowest mortality. Cell functionality is also conserved.

The 4D-Nucleofector™ System was developed to offer advanced performance, flexibility and convenience for cell transfection purposes. The system has a modular architecture that allows seamless expansion of the system: it comprises a base core unit and functional units to suit your application of interest. Lonza provides over 160 optimized protocols and transfection kits.

The 4D-Nucleofector™ X-Unit is the most flexible unit and supports nucleofection of various cell numbers in different formats, as it is equipped with a strip position (16-well strip format) and 2 cuvette positions. This allows you to be flexible in the transfection of your cell type of interest, using a smaller volume or a higher volume. The X-Unit features positions for 20µl Nucleocuvette™ strips (for low cell numbers down to 2 x 10^4) and 100 μl single Nucleocuvette™ (for cell numbers up to 2 x 10^7). Different Nucleofection Vessels allow for flexible throughput from 1 to 16 samples.  The system provides seamless transfer of conditions between different Nucleofection Vessels: same protocol can be used for use of the strips and the cuvettes.

At BioTether Sciences we use the Lonza Nucleofector 4D system for delivery of plasmids, siRNA oligonucleotides, gRNA, RNP and proteins to a variety of cell types. The system allows for high efficiency delivery of CRISPR/Cas9 RNPs for gene modification. The use of RNPs can reduce off target editing and in hard to transfect cell lines and primary cells, reduce toxicity. For instance, we use the Nucleofector 4D for gene knock-out studies. The high efficiency delivery helps get us a step closer to obtaining a pure population of knock-out cells, especially when combined with our cell separation technologies. 

Biomaterials and polymers are increasingly combined with drugs and biologics. Regenerative products may have medical device–based scaffolding and may be treated as biologics, reflecting the cell and tissue components. Therefore, biomaterials, medical device components, and 3-D printed materials need to be fully characterized for biocompatibility. Biological reactivity tests (see USP <87> and ISO 10993-5 guidances) are designed to determine the biocompatibility of mammalian cell cultures following contact with elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from the materials under test.

For medical devices, all biocompatibility and efficacy testing occurs prior to any clinical testing. Being aware of and applying the proper testing standards to development and testing of the device can facilitate entry into the desired national or global market place.  Medical devices are characterized using cell culture assay to assess the biocompatibility of a material or extract through the use of mammalian cells. These techniques provide an excellent way to screen materials prior to expensive animal testing.

Per the USP <87> monograph, there are three cytotoxicity tests commonly used for medical devices. The direct contact test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very low- or high-density materials that could cause mechanical damage to the cells. Reactivity of the test sample is indicated by changes in morphology, membrane degeneration and lysis of cells around the test material.

The agar diffusion test is designed for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens.

The MEM Elution test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the specimens at physiological or non-physiological temperature. This test may use different extraction media and extraction conditions to test devices according to actual use conditions or to exaggerate those conditions.  After preparation, the extracts are transferred onto a layer of cells (typically mouse fibroblast L-929 cells).  Following incubation, the cells are examined microscopically for morphology and lysis of the cells.

Recent regulations (ANSI/AAMI/ISO 10993-5:2009) on biocompatibility for devices state that the three qualitative cytotoxicity tests are appropriate for screening purposes, but that quantitative evaluation is recommended.  There are several quantitative cell viability assays available such as colorimetric MTT assay, FL staining for live/dead cells, or luciferase based cell viability testing. Our laboratory has cell imaging capabilities that include multi-color Fluorescence (using the Thermo Evos cell imaging platform) or plate based quantitative spectrophotometric analysis using UV/Vis, FL, and luminescence detection (using Molecular Devices platforms). Cell cytotoxicity assays are just one way we can characterize your medical device, or biopharmaceutical product.

Hundreds of millions of dollars are available to small biotechnology and medical device businesses to fight COVID-19. The money is in the form of non-dilutive grants, contracts,  and also in exchange for equity positions in your company. For example start-up incubators may offer small seed funds of $25,000-$50,000 in exchange for residency, mentorship and an equity position in your company. Other funding includes large multi-year grants of $5,000,000 or more offered by the Department of Defense. The monies may bring forth the best and brightest ideas from biotechnology and medical device start-ups and other small businesses. These funded innovations could be critical in the fight against COVID-19 and other infectious diseases. The solutions to the current crisis may help us improve healthcare and disaster readiness for years to come.

The money is coming from public and private sources. For-profit and non-profit institutions. Military and civilian agencies. These funding sources accelerate the development and availability of transformative technologies and approaches to protect Americans from health security threats. Here is a partial list and links to some of the Health and Human Services and Department of Defense  related funding agencies: NIH-SBIR (https://www.sbir.gov/) , CDC (https://www.cdc.gov/) , BARDA (https://www.medicalcountermeasures.gov/barda/) , DARPA (https://www.darpa.mil/), MTEC (https://www.mtec-sc.org/) , MCDC https://www.medcbrn.org/).

Department of Defence (DoD) Consortiums like MTEC and MCDC, facilitate DoD and industrial relationships and funding agreements . Today they are advancing countermeasures to COVID-19 and other infectious diseases. The mission is to protect the population and improve war fighting capabilities.  These agencies also understand that the funding helps small businesses survive in tough times and the military has a clear need to promote resilience of the defense industrial base during the COVID-19 pandemic. Biotechnology solutions, point of care medical devices, and therapeutics have become the armaments of the war on pandemics.

Philanthropic organizations such as the Bill and Melinda Gates Foundation and the Chan Zuckerberg Initiative are offering funds to fight COVID-19 and care for the sick. These types of organizations can provide expert advice and support to start-ups to bring their solutions to the COVID-19 induced healthcare crisis. 

Several start-up Incubators such as SOSventures/IndieBio, QB3, J&J Innovation Centers are offering seed funds ranging from $25,000 to $250,000 in exchange for an equity stake in the venture. The COVID-19 specific offerings have an urgency to them not seen in the past.The announcements are occuring at a fast clip in March and April. Often applicants are given days to weeks to respond with a technical proposal and detailed budget. Often the product or idea must be completed and ready for deployment  in 3-12 months. The areas of interest include Point of Care Diagnostics, Prophylactics, Ready-to-Go Therapeutics, Medical Devices, Protective Gear

Computer Modeling, Disease Tracking, and AI for Drug Discovery.

BioTether Sciences is proud to be a member of Medical Technology Enterprise Consortium, (MTEC), the NIH-Small Business Innovative Research (SBIR) program, and the State and Federal systems for award management (eProcure, and betaSAM.gov). We are developing technologies for diagnostics, prophylactic and therapeutics to fight COVID-19. Our technologies exploit high affinity interactions between target-receptor, antigen-antibody and other ways macromolecules interact.

(Created using information from the CDC.gov website)

The CDC works closely with state and local public health departments, travel industry partners, and others to identify and test people who may be infected with COVID-19. There are  several different laboratory tests to detect COVID-19 infection. The testing strategy is based on experience from the SARS and MERS outbreaks.

In general, these lab tests fall into two categories:

• Molecular tests, which look for evidence of active infection; and
• Serology tests, which look for previous infection by detecting antibodies to COVID-19. Serology tests are for surveillance or investigational purposes and not for diagnostic purposes.

Molecular Tests

Molecular tests are used to diagnose active infection (presence of COVID-19) in people who are thought to be infected based on their clinical symptoms and having links to places where COVID-19 has been reported.

• Real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays are molecular tests that can be used to detect viral RNA in clinical samples. CDC’s current case definition for laboratory confirmation of COVID-19 infection requires a positive rRT-PCR result for at least two specific genomic targets. Sequencing may also be used as a confirmatory test.
• Increasingly laboratories in the United States are approved to test for COVID-19 by using an rRT-PCR assay developed by CDC. This test is done under authority of an Emergency Use Authorization because there are no FDA-cleared/approved tests available for this purpose in the United States.
• The success of rRT-PCR testing depends on several factors, including the experience and expertise of laboratory personnel, laboratory environment (e.g., avoidance of contamination), and the type and condition of specimens being tested. For this rRT-PCR assay, CDC recommends collecting multiple specimens, including lower (bronchalveolar lavage, sputum and tracheal aspirates) and upper (e.g., nasopharyngeal and oropharyngeal swabs) respiratory samples, serum, and stool specimens.
• CDC considers a person under investigation to be negative for active COVID-19 infection following one negative rRT-PCR test on the recommended specimens. Since a single negative result does not completely rule out infection, in some circumstances additional specimens may be tested.
• CDC considers a known the patient to be negative for active COVID-19 infection following two consecutive negative rRT-PCR tests on all specimens.

Serology Tests

Serology testing is used to detect previous infection (antibodies to COVID-19) in people who may have been exposed to the virus. Antibodies are proteins produced by the body’s immune system to attack and kill viruses, bacteria, and other microbes during infection. The presence of antibodies to COVID-19 indicates that a person had been previously infected with the virus and developed an immune response.

• Evidence to date suggests there may be a broader range of COVID-19 disease than was initially thought. For this reason, public health scientists are working to learn more about how the virus is transmitted. One way to do this is through voluntary testing of blood samples from people who had close contact with people known to have COVID-19.
• CDC has a two-phase approach for serology testing, using two screening tests and one confirmatory test to detect antibodies to MERS-CoV.
• ELISA, or enzyme-linked immunosorbent assay, is a screening test used to detect the presence and concentration of specific antibodies that bind to a viral protein.
• The microneutralization assay is a highly specific confirmatory test used to measure neutralizing antibodies, or antibodies that can neutralize virus. This method is considered a gold standard for detection of specific antibodies in serum samples. However, compared with the ELISA, the microneutralization assay is labor-intensive and time-consuming, requiring at least 5 days before results are available.
• If a clinical sample is positive by either ELISA, and positive by microneutralization, the specimen is determined to be confirmed positive.
• If a clinical sample is positive by both ELISAs, and negative by microneutralization, the sample is determined to be indeterminate.
• If a clinical sample is positive by only one ELISA, and negative by microneutralization, the sample is determined to be negative.
• If a clinical sample is negative by both ELISAS, the sample is determined negative.
• In the end, a final determination of a confirmed positive serology result requires a positive ELISA test and confirmation by microneutralization assay.