Monthly Archives: July 2016

3DCellMaker for bio-relevant preclinical testing in conventional microplate assays

 

The PolySciTech product 3DCellMaker (www.3dcellmaker.com) provides a thermogel based platform which allows tumor cells to grow into three-dimensional shapes and structures which have significantly more bio-relevance than 2-dimensional cell monolayers. Multiple studies have shown this to be the case particularly in regards to the micro-environmental difference between the central ‘core’ portion of tumors as compared to the surface environment. This difference is not represented in 2D monolayers and, often, contributes to cancer having resistance to various drugs and chemotherapeutic strategies. Recently, researchers have published regarding protocols for assays of 3D spheroids in microplates by conventional microplate methods. Because 3DCellMaker allows for 3D cell growth in any plasticware or glassware regardless of the container’s dimension, it is well adapted for these types of assays. Read more about these assays here: Vinci, Maria, Sharon Gowan, Frances Boxall, Lisa Patterson, Miriam Zimmermann, Cara Lomas, Marta Mendiola, David Hardisson, and Suzanne A. Eccles. “Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.” BMC biology 10, no. 1 (2012): 1. http://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-10-29

“Abstract: Background: There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results: To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease. Conclusions: We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds. Keywords: 3D angiogenesis drug response high throughput invasion migration tumor spheroids”

PLGA from PolySciTech used in development of Genipin eluting structure for repair of connective tissue

 

PolySciTech division of Akina, Inc. (www.polyscitech.com) provides a wide array of biodegradable polymers including PLGA. Recently, PLGA (Polyvivo AP081, PLGA 5000-10,000 acid endcap) from PolySciTech was utilized by researchers at the University of Kentucky to generate genipin releasing sutures for tendon repair. This research may hold promise for improved tendon repair after trauma or surgery. Read more: Sundararaj, Sharath, Paul Slusarewicz, Matt Brown, and Thomas Hedman. “Genipin crosslinker releasing sutures for improving the mechanical/repair strength of damaged connective tissue.” Journal of Biomedical Materials Research Part B: Applied Biomaterials (2016). http://onlinelibrary.wiley.com/doi/10.1002/jbm.b.33753/full

“Abstract: The most common mode of surgical repair of ruptured tendons and ligaments involves the use of sutures for reattachment. However, there is a high incidence of rerupture and repair failure due to pulling out of the suture material from the damaged connective tissue. The main goal of this research was to achieve a localized delivery of crosslinking agent genipin (GP) from rapid-release biodegradable coatings on sutures, for strengthening the repair of ruptured connective tissue. Our hypothesis is that GP released from the suture coating will lead to exogenous crosslinking of native connective tissue resulting in beneficial effects on clinically relevant mechanical parameters such as tear resistance, tissue strength, and energy required to rupture the tissue (toughness). Sutures were successfully coated with a biodegradable polymer layer loaded with the crosslinking agent genipin, without compromising the mechanical properties of the suture. The rapid-release of genipin was achieved under both in vitro and ex vivo conditions. Exogenous crosslinking using these genipin releasing sutures was demonstrated using equine tendons. The tendons treated with genipin releasing sutures showed significant improvement in failure load, energy required for pull-out failure, and stiffness. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016. Keywords: controlled release; bioresorbable; coating(s); connective tissue; drug delivery/release”

3DCellMaker Poster presentation at Controlled Release Society 2016 Annual Meeting

 

3DCellMaker (www.3DCellMaker.com) is a thermogelling cell-growth media modifier which allows for culturing cells in 3D structures without requiring specialized plasticware or equipment. At the 2016 CRS meeting, Justin Hadar presented a research poster generated from a collaboration between Akina, Inc. and Purdue University Department of Basic Medical Science’s Professor Sophie Leliévre. For this poster, multiple cell-lines were cultured as well as tests of co-culture between MCF-7 cells with associated fibroblasts and e-cadherin staining to establish formation of cell-to-cell interactions. You can see the full poster and supplemental data here (http://www.3dcellmaker.com/research/). Abstract attached below:

“ABSTRACT: 3DCellMakers: Thermogelling Polymers for 3D Cell Culture, J. Hadar1, J. Garner1 S. Skidmore1, H. Park1, K. Park1,2, B. Han2, F. Atrian2, S.A. Lelièvre2  1Akina, Inc. West Lafayette, IN 47906 USA 2Purdue University West Lafayette, IN 47907 USA jh@akinainc.com Purpose: The goal of this study is to synthesize polymers that provide consistent, reproducible environments for cells to form tumor structures, resulting in more representative drug transport and therapeutic characteristics relevant to clinical applications.  Certain types of inverse thermogelling polymers allow tumor cells as well as non-disease state cells to form three-dimensional (3D) spheroid-like structures with characteristic features that are not observed in the same cells cultured in 2D. The thermogelling property allows mixing cells with polymer solution at room temperature before forming a transparent gel at 37 °C. Methods:  Ethylene oxide sterilized thermogels were dissolved in cell culture medium which consisted of DMEM/F12 + GlutaMAX™ basal medium supplemented with 5% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).  The volume of medium was adjusted for the desired % (w/v) hydrogel, and dissolution was done at 2-8 °C for two days.  Multiple cell lines were used to conduct the experiments. In one approach either breast cancer MCF-7 cells cocultured with human fibroblasts CCD-1068SK or human liver carcinoma HEP G2 cells were mixed with cold hydrogel, and the mixtures were transferred into   a flat bottom polystyrene multiwall plate (uncoated) and incubated at 37 °C. In another approach, the hydrogel solution was prewarmed in polystyrene multiwall plates and the cell suspension was added on top of the solidified gel. At predetermined time points, pictures of cell cultures were taken. During the culture period, the medium was replaced every 48 to 72 hours. Additionally, triple negative breast cancer T4-2 cells typically cultured in a serum-free medium with known additives were seeded onto 100 ul of 3DCellMaker gel per well in a 12-well plate and images were taken after two days of culture.  Results: Three promising polymers for 3D cell culture were identified, and they were named “3DCellMakers”. They include poloxamer-hexamethylene diisocyanate poly(ester-urethane) (PEU), stearate modified methyl cellulose, and poloxamer-methylene diphenyl diisocyanate PEU. In general, seeding the cells onto the prewarmed gels allow 3D structures to form quickly (1~4 days), while mixing the cells directly with cold thermogel solution before heating to gel typically yielded tumors at a later time and of smaller size.  Tumors ranged in size from 40 um to 200 um (Fig. 1).  T4-2 cells that are particularly sensitive to their environment for tumor formation also formed 3D structures in less than 48 hours (Fig. 2). Conclusions: The 3DCellMakers have potential to provide an effective, inexpensive, and easy method for generating 3D multicellular structures.  The new thermogelling polymers provide a new avenue of increased productivity in cell biology research for which multicellular 3D structure formation is critical, e.g., studying the efficacy of various drugs and drug delivery systems for treating tumors.  The ability to co-culture cancer cells with fibroblasts in 3D provides an interesting avenue to study important aspects of the tumor microenvironment, especially if it can be combined with microfluidic devices or high throughput screening systems. Additionally, the new thermogelling polymers allow T4-2 invasive cancer cells, which are known to be sensitive to the microenvironmental conditions and are cultured under serum-free conditions, to thrive. This preliminary result suggests that the 3DCellMaker has potential for broad use. The possibility to culture cells with reproducible and known medium conditions will make easier the study of the effect of specific components on tumor growth.”

PLGA-diol precursor from PolySciTech used in biodegradable polyurethane development

PolySciTech Division of Akina, Inc. (www.polyscitech.com) provides a wide variety of research polymers. This includes PLGA di-alcohol precursors such as HO-PLGA-TEG-PLGA-OH (Polyvivo AK076). The dialcohol units on these polymers can react with isocyanate groups to form urethane linkages. Since the polymer is difunctionalized, it joins into the forming chain of a polyurethane and acts as a chain extender linked di-isocyanates together. Although typical polyurethanes are not biodegradable, the incorporation of PLGA into the polyurethane backbone in this way renders the polymer as a degradable matrix useful for a wide array of applications. Read more about this exciting technology here: Blakney, Anna K., Felix Simonovsky, Ian T. Suydam, Buddy D. Ratner, and Kim A. Woodrow. . “Rapidly Biodegrading PLGA-Polyurethane Fibers for Sustained Release of Physicochemically Diverse Drugs”. ACS Biomaterials Science & Engineering 2016 (http://pubs.acs.org/doi/abs/10.1021/acsbiomaterials.6b00346)

“Abstract: Sustained release of physicochemically diverse drugs from electrospun fibers remains a challenge and precludes the use of fibers in many medical applications. Here we synthesize a new class of polyurethanes with PLGA moieties that degrade faster than polyurethanes based on polycaprolactone. The new polymers, with varying hard to soft segment ratios and fluorobenzene pendant group content, were electrospun into nanofibers and loaded with four topically relevant but physicochemically diverse small molecule drugs. Polymers were characterized using GPC, XPS and 19F NMR. The size and morphology of electrospun fibers were visualized using SEM, and drug/polymer compatibility and drug crystallinity were evaluated using DSC. We measured the in vitro release of physicochemically diverse drugs, and polymer degradation and cytotoxicity of biodegradation products were evaluated in cell culture. We show that these newly synthesized PLGA-based polyurethanes degrade up to 65-80% within four weeks, and are cytocompatible in vitro. The resulting drug-loaded electrospun fibers were amorphous solid dispersions. We found that increasing the hard to soft segment ratio of the polymer enhances the sustained release of positively charged drugs, while increasing the fluorobenzene pendant content caused more rapid release of some drugs. Increasing the hard segment or fluorobenzene pendant content of segmented polyurethanes containing PLGA moieties allows for modulation of physicochemically diverse drug release from electrospun fibers, while maintaining a biologically relevant biodegradation rate.”

pH sensitive Aquagel from Akina, Inc. used for strain-based pH sensor development

PolySciTech division of Akina, Inc (www.polyscitech.com) provides a wide variety of research products including superporous hydrogels developed using Akina’s Aquagel technology originally spun-off from Purdue University under Kinam Park’s lab. One of the materials marketed by this route is pH sensitive aquagel that deswells in acidic conditions (https://akinainc.com/polyscitech/products/aquagel/AquaGel-pH.php). Recently, this aquagel was combined with a thin PDMS membrane and an advanced laser detection system to create a pH sensor based on hydrogel strain. This research holds the potential for developing robust micro-sensors for small-scale applications such as detection of local pH changes in biological systems. Read more about this here: Choi, Jae-Hyuck, You-Shin No, Jae-Pil So, Jung Min Lee, Kyoung-Ho Kim, Min-Soo Hwang, Soon-Hong Kwon, and Hong-Gyu Park. “A high-resolution strain-gauge nanolaser.” Nature communications 7 (2016). http://www.nature.com/ncomms/2016/160512/ncomms11569/full/ncomms11569.html?WT.ec_id=NCOMMS-20160518

“Abstract: Interest in mechanical compliance has been motivated by the development of flexible electronics and mechano-sensors. In particular, studies and characterization of structural deformation at the fundamental scale can offer opportunities to improve the device sensitivity and spatiotemporal response; however, the development of precise measurement tools with the appropriate resolution remains a challenge. Here we report a flexible and stretchable photonic crystal nanolaser whose spectral and modal behaviours are sensitive to nanoscale structural alterations. Reversible spectral tuning of ~26 nm in lasing wavelength, with a sub-nanometer resolution of less than ~0.6 nm, is demonstrated in response to applied strain ranging from −10 to 12%. Instantaneous visualization of the sign of the strain is also characterized by exploring the structural and corresponding modal symmetry. Furthermore, our high-resolution strain-gauge nanolaser functions as a stable and deterministic strain-based pH sensor in an opto-fluidic system, which may be useful for further analysis of chemical/biological systems.”

PEG-PLA and oligomeric PLA used for paclitaxel delivery

PolySciTech division of Akina, Inc. (www.polyscitech.com) provides a wide array of biodegradable research products including short-chain PLA and PEG-PLA copolymers. Recently, researchers at the University of Wisconsin-Madison conjugated short-chain PLA to paclitaxel in order to form a prodrug (e.g. a molecule which degrades in the human body to generate a medicinal molecule). Subsequently, they loaded this prodrug into PEG-PLA nanocarriers. This system was tested in mice and it was found that the tumors (A549 human lung cancer) actually shrunk and regressed wheras loose PTX only served to only delay tumor growth. This research holds promise for improved chemotherapeutic outcomes. Read more: Tam, Yu Tong, Jieming Gao, and Glen S. Kwon. “Oligo (lactic acid) n-paclitaxel prodrugs for poly (ethylene glycol)-block-poly (lactic acid) micelles: Loading, release and backbiting conversion for anticancer activity.” Journal of the American Chemical Society (2016). http://pubs.acs.org/doi/abs/10.1021/jacs.6b03995

“Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles are nanocarriers for poorly water-soluble anticancer agents and have advanced paclitaxel (PTX) to humans due to drug solubilization, biocompatibility, and dose escalation. However, PEG-b-PLA micelles rapidly release PTX, resulting in widespread biodistribution and low tumor exposure. To improve delivery of PTX by PEG-b-PLA micelles, monodisperse oligo(l-lactic acid), o(LA)8 or o(LA)16, has been coupled onto PTX at the 7-OH position, forming ester prodrugs: o(LA)8-PTX and o(LA)16-PTX, respectively. As expected, o(LA)n-PTX was more compatible with PEG-b-PLA micelles than PTX, increasing drug loading from 11 to 54%. While in vitro release of PTX was rapid, resulting in precipitation, o(LA)n-PTX release was more gradual: t1/2 = 14 and 26 h for o(LA)8-PTX and o(LA)16-PTX, respectively. Notably, o(LA)8-PTX and o(LA)16-PTX in PEG-b-PLA micelles resisted backbiting chain end scission, based on reverse-phase HPLC analysis. By contrast, o(LA)8-PTX and o(LA)16-PTX degraded substantially in 1:1 acetonitrile:10 mM PBS, pH 7.4, at 37 °C, generating primarily o(LA)2-PTX. The IC50 value of o(LA)2-PTX was ∼2.3 nM for A549 human lung cancer cells, equipotent with PTX in vitro. After weekly IV injections at 20 mg/kg as PEG-b-PLA micelles, o(LA)8-PTX induced tumor regression in A549 tumor-bearing mice, whereas PTX delayed tumor growth. Surprisingly, o(LA)8-PTX caused less toxicity than PTX in terms of change in body weight. In conclusion, o(LA)n acts as a novel promoiety, undergoing backbiting conversion without a reliance on metabolizing enzymes, and o(LA)n-PTX improves PTX delivery by PEG-b-PLA micelles, providing a strong justification for clinical evaluation.”

PolymBlend investigated for generating Biosensor of serotonin in serum

PolySciTech division of Akina, Inc. (www.polyscitech.com) provides a wide array of research products both from PolySciTech itself as well as distributed products. One of the distributed products available from PolySciTech is PolymBlend material used for electrospinning (https://akinainc.com/polyscitech/products/polymblend/index.php) recently, this material was utilized for the generation of a fluorescence based biosensor. Read more here: Ramon-Marquez, Teresa, Antonio L. Medina-Castillo, Alberto Fernandez-Gutierrez, and Jorge F. Fernandez-Sanchez. “A novel optical biosensor for direct and selective determination of serotonin in serum by Solid Surface-Room Temperature Phosphorescence.” Biosensors and Bioelectronics 82 (2016): 217-223. http://www.sciencedirect.com/science/article/pii/S0956566316302846

“Abstract: This paper describes a novel biosensor which combines the use of nanotechnology (non-woven nanofibre mat) with Solid Surface-Room Temperature Phosphorescence (SS-RTP) measurement for the determination of serotonin in human serum. The developed biosensor is simple and can be directly applied in serum; only requires a simple clean-up protocol. Therefore it is the first time that serotonin is analysed directly in serum with a non-enzymatic technique. This new approach is based on the covalent immobilization of serotonin directly from serum on a functional nanofibre material (Tiss®-Link) with a preactivated surface for direct covalent immobilization of primary and secondary amines, and the subsequent measurement of serotonin phosphorescent emission from the solid surface. The phosphorescent detection allows avoiding the interference from any fluorescence emission or scattering light from any molecule present in the serum sample which can be also immobilised on the nanofibre material. The determination of serotonin with this SS-RTP sensor overcomes some limitations, such as large interference from the matrix and high cost and complexity of many of the methods widely used for serotonin analysis. The potential applicability of the sensor in the clinical diagnosis was demonstrated by analysing serum samples from seven healthy volunteers. The method was validated with an external reference laboratory, obtaining a correlation coefficient of 0.997 which indicates excellent correlation between the two methods. Keywords: Nanotechnology; Tiss®-Link; SS-RTP; Nanofibre mat; Serum; Phosphorescence; Serotonin; Protein clean-up”

PLGA from PolySciTech used for creating pH sensitive nanofiber scaffold for live-cell pH analysis

PolySciTech division of Akina, Inc. (www.polyscitech.com) provides a wide array of biodegradable polymers including poly(lactide-co-glycolide). Recently, PolySciTech PLGA 85:15 LA:GA, ester capped, Mn 100-200kDa (PolyVivo Catalog # AP075) was used to form the polymeric base of an electrospun mesh which incorporated fluorophores and ionic additives. This research holds promise for developing a scaffold that allows for growth of cells and monitoring of their pH condition locally for improved research and understanding. Read more: Di, Wenjun, Ryan S. Czarny, Nathan A. Fletcher, Melissa D. Krebs, and Heather A. Clark. “Comparative Study of Poly (ε-Caprolactone) and Poly (Lactic-co-Glycolic Acid)-Based Nanofiber Scaffolds for pH-Sensing.” Pharmaceutical Research (2016): 1-12. http://link.springer.com/article/10.1007/s11095-016-1987-0

“Abstract: Purpose: This study aims to develop biodegradable and biocompatible polymer-based nanofibers that continuously monitor pH within microenvironments of cultured cells in real-time. In the future, these fibers will provide a scaffold for tissue growth while simultaneously monitoring the extracellular environment. Methods: Sensors to monitor pH were created by directly electrospinning the sensor components within a polymeric matrix. Specifically, the entire fiber structure is composed of the optical equivalent of an electrode, a pH-sensitive fluorophore, an ionic additive, a plasticizer, and a polymer to impart mechanical stability. The resulting poly(ε-caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA) based sensors were characterized by morphology, dynamic range, reversibility and stability. Since PCL-based nanofibers delivered the most desirable analytical response, this matrix was used for cellular studies. Results: Electrospun nanofiber scaffolds (NFSs) were created directly out of optode material. The resulting NFS sensors respond to pH changes with a dynamic range centered at 7.8 ± 0.1 and 9.6 ± 0.2, for PCL and PLGA respectively. NFSs exhibited multiple cycles of reversibility with a lifetime of at least 15 days with preservation of response characteristics. By comparing the two NFSs, we found PCL-NFSs are more suitable for pH sensing due to their dynamic range and superior reversibility. Conclusion: The proposed sensing platform successfully exhibits a response to pH and compatibility with cultured cells. NSFs will be a useful tool for creating 3D cellular scaffolds that can monitor the cellular environment with applications in fields such as drug discovery and tissue engineering. KEY WORDS: electrospinning nanofibers pH detection poly(lactic-co-glycolic acid) poly(ε-caprolactone)”

Diacrylated biodegradable copolymers for photo-initiated 3D printing available from PolySciTech

PolySciTech division of Akina, Inc (www.polyscitech.com) provides a wide array of biodegradable polymers including vinyl modified block copolymers which can be used as macromers. Examples of this include PLGA-PEG-PLGA-diacrylate, PLA-diacrylate, and others available through our reactive intermediate series (PolyVivo AI***). These polymers can be dissolved in an appropriate solvent or blended in a melt along with an appropriate photo-initiator (Irgacure or other) and 3D printed under illuminated conditions to create a chemically crosslinked structure. This soft-printing technique at low temperature conditions allows for the potential of printing living cells or other biologics directly into the printed matrix.  Historically, researchers have been limited to very few commercially available magromers, such as poly(ethylene glycol) diacrylate, for this application. This macromere prints well but has limited cell interaction and poorly controlled degradability. The use of biodegradable macromers with polyester blocks can allow for more controlled degradability. A recent review article highlights the potential for printing living cells. Read more: Park, Jeong Hun, Jinah Jang, Jung-Seob Lee, and Dong-Woo Cho. “Three-Dimensional Printing of Tissue/Organ Analogues Containing Living Cells.” Annals of biomedical engineering (2016): 1-15. http://link.springer.com/article/10.1007/s10439-016-1611-9

“Abstract: The technical advances of three-dimensional (3D) printing in the field of tissue engineering have enabled the creation of 3D living tissue/organ analogues. Diverse 3D tissue/organ printing techniques with computer-aided systems have been developed and used to dispose living cells together with biomaterials and supporting biochemicals as pre-designed 3D tissue/organ models. Furthermore, recent advances in bio-inks, which are printable hydrogels with living cell encapsulation, have greatly enhanced the versatility of 3D tissue/organ printing. Here, we introduce 3D tissue/organ printing techniques and biomaterials that have been developed and widely used thus far. We also review a variety of applications in an attempt to repair or replace the damaged or defective tissue/organ, and develop the in vitro tissue/organ models. The potential challenges are finally discussed from the technical perspective of 3D tissue/organ printing. Keywords: 3D tissue/organ printing Bio-inks 3D tissue/organ analogues Tissue engineering and regenerative medicine 3D in vitro tissue/organ models.”

Meet PolySciTech at CRS Annual Meeting Seattle July 17-20th, Booth 506

Representatives from PolySciTech division of Akina, Inc. (John Garner and Justin Hadar) (www.polyscitech.com) will be at controlled release society (CRS) meeting in Seattle this year July 17-20th. Drop by our booth #506 for a chat and a free t-shirt (supplies limited). Also check out our scientific posters regarding PLGA analysis techniques (Poster # 239 “Assay of PLGA Properties in Parenteral Depot Formulations”) and three-dimensional cell-growth medium (Poster # 608 “3D CellMakers: Thermogelling Polymers for 3D Cell Culture”). You can learn more about the CRS meeting here http://www.controlledreleasesociety.org/meetings/annual/Pages/default.aspx