<img height="1" width="1" src="https://www.facebook.com/tr?id=1582471781774081&amp;ev=PageView &amp;noscript=1">
  • Menu
  • crown-logo-symbol-1-400x551

Find it Quickly

Get Started

Select the option that best describes what you are looking for

  • Services
  • Models
  • Scientific Information

Search Here For Services

Click Here to Start Over

Search Here For Models

Click Here to Start Over

Search Here For Scientific Information

Click Here to Start Over

In Vitro

Boost oncology drug discovery with XenoBase®, featuring the largest cell line selection and exclusive 3D organoid models. Benefit from OrganoidXplore™ and OmniScreen™ for rapid, in-depth analysis.

Learn More

In Vivo

Enhance drug development with our validated in vivo models, in vitro/ex vivo assays, and in silico modeling. Tailored solutions to optimize your candidates.

Learn More

Tissue

Experience ISO-certified biobanking quality. Access top biospecimens from a global clinical network, annotated by experts for precise research.

Learn More

Biomarkers and Bioanalysis

Leverage our global labs and 150+ scientists for fast, tailored project execution. Benefit from our expertise, cutting-edge tech, and validated workflows for reliable data outcomes.

Learn More

Data Science and Bioinformatics

Harness your data and discover biomarkers with our top bioinformatics expertise. Maximize data value and gain critical insights to accelerate drug discovery and elevate projects.

Learn More

KRAS

Accelerate innovative cancer treatments with our advanced models and precise drug screening for KRAS mutations, efficiently turning insights into clinical breakthroughs.

Learn More

EGFR

Advance translational pharmacology with our diverse pre-clinical models, robust assays, and data science-driven biomarker analysis, multi-omics, and spatial biology.

Learn More

Drug Resistance

Our suite integrates preclinical solutions, bioanalytical read-outs, and multi-omics to uncover drug resistance markers and expedite discovery with our unique four-step strategy.

Learn More

Patient Tissue

Enhance treatments with our human tumor and mouse models, including xenografts and organoids, for accurate cancer biology representation.

Learn More

Target to Lead Selection for ADCs and Biologics

Journey through the drug discovery pipeline from target discovery to in vivo pharmacology. Take advantage of the Largest biobank of patient-derived models, Model development capabilities, Cell-based assays for Screening ADCs, and advanced downstream analysis.

Learn More

Bioinformatics

Apply the most appropriate in silico framework to your pharmacology data or historical datasets to elevate your study design and analysis, and to improve your chances of clinical success.

Learn More

Biomarker Analysis

Integrate advanced statistics into your drug development projects to gain significant biological insight into your therapeutic candidate, with our expert team of bioinformaticians.

Learn More

CRISPR/Cas9

Accelerate your discoveries with our reliable CRISPR solutions. Our global CRISPR licenses cover an integrated drug discovery platform for in vitro and in vivo efficacy studies.

Learn More

Genomics

Rely on our experienced genomics services to deliver high quality, interpretable results using highly sensitive PCR-based, real-time PCR, and NGS technologies and advanced data analytics.

Learn More

In Vitro High Content Imaging

Gain more insights into tumor growth and disease progression by leveraging our 2D and 3D fluorescence optical imaging.

Learn More

Mass Spectrometry-based Proteomics

Next-generation ion mobility mass spectrometry (MS)-based proteomics services available globally to help meet your study needs.

Learn More

Ex Vivo Patient Tissue

Gain better insight into the phenotypic response of your therapeutic candidate in organoids and ex vivo patient tissue.

Learn More

Spatial Multi-Omics Analysis

Certified CRO services with NanoString GeoMx Digital Spatial Profiling.

Learn More

Biomarker Discovery

De-risk your drug development with early identification of candidate biomarkers and utilize our biomarker discovery services to optimize clinical trial design.

Learn More

DMPK Services

Rapidly evaluate your molecule’s pharmaceutical and safety properties with our in vivo drug metabolism and pharmacokinetic (DMPK) services to select the most robust drug formulations.

Learn More

Efficacy Testing

Explore how the novel HuGEMM™ and HuCELL™ platforms can assess the efficacy of your molecule and accelerate your immuno-oncology drug discovery programs.

Learn More

Laboratory Services

Employ cutting-edge multi-omics methods to obtain accurate and comprehensive data for optimal data-based decisions.

Learn More

Pharmacology & Bioanalytical Services

Leverage our suite of structural biology services including, recombinant protein expression and protein crystallography, and target validation services including RNAi.

Learn More

Screens

Find the most appropriate screen to accelerate your drug development: discover in vivo screens with MuScreen™ and in vitro cell line screening with OmniScreen™.

Learn More

Toxicology

Carry out safety pharmacology studies as standalone assessments or embedded within our overall toxicological profiling to assess cardiovascular, metabolic and renal/urinary systems.

Learn More

Preclinical Consulting Services

Learn more about how our consulting services can help to support your journey to the clinic.

Learn More

Our Company

Global CRO in California, USA offering preclinical and translational oncology platforms with high-quality in vivo, in vitro, and ex vivo models.

Learn More

Our Purpose

Learn more about the impact we make through our scientific talent, high-quality standards, and innovation.

Learn More

Our Responsibility

We build a sustainable future by supporting employee growth, fostering leadership, and exceeding customer needs. Our values focus on innovation, social responsibility, and community well-being.

Learn More

Meet Our Leadership Team

We build a sustainable future by fostering leadership, employee growth, and exceeding customer needs with innovation and social responsibility.

Learn More

Scientific Advisory Board

Our Scientific Advisory Board of experts shapes our strategy and ensures top scientific standards in research and development.

Learn More

News & Events

Stay updated with Crown Bioscience's latest news, achievements, and announcements. Check our schedule for upcoming events and plan your visit.

Learn More

Career Opportunities

Join us for a fast-paced career addressing life science needs with innovative technologies. Thrive in a respectful, growth-focused environment.

Learn More

Scientific Publications

Access our latest scientific research and peer-reviewed articles. Discover cutting-edge findings and insights driving innovation and excellence in bioscience.

Learn More

Resources

Discover valuable insights and curated materials to support your R&D efforts. Explore the latest trends, innovations, and expertly curated content in bioscience.

Learn More

Blogs

Explore our blogs for the latest insights, research breakthroughs, and industry trends. Stay educated with expert perspectives and in-depth articles driving innovation in bioscience.

Learn More

  • Platforms
  • Target Solutions
  • Technologies
  • Service Types

Developing Effective Dosing Strategies by Leveraging 3D In Vitro Models

Drug attrition rates remain high, and this has been attributed in part to the poor translatability of current preclinical data. Models that have higher predictivity of patient response are needed.

In this post, we explore how developing effective dosing strategies by leveraging 3D in vitro models (e.g., organoids, cell lines grown in 3D, and ex vivo patient tissue (EVPT)), that more closely recapitulate the complex in vivo environment, can be used for making better decisions, including single or multi-drug combinations.

Why Use 3D In Vitro Models for Drug Screening Studies?

To overcome the poor translatability of current preclinical data, there is an immediate need for more advanced preclinical models beginning at the earliest steps of drug discovery (i.e., high-throughput drug screening (HTS)) so that the complexities of human disease are better recapitulated, allowing for better decisions earlier in drug discovery. Over the recent past, amazing technological advances have led to alternatives to traditional 2D monolayer culture systems, such as 3D in vitro organoids and EVPT platforms that more closely recapitulate the complex in vivo environment, and thus, show high predictivity of patient response.

Furthermore, using advanced 3D in vitro models has the potential to revolutionize the traditional linear drug-screening workflow, which has typically identified a lead compound first and then as a separate step, seeks to identify the target patient population. By using a matrix HTS workflow, multiple patient-relevant models can be used simultaneously to reflect patient characteristics at not only the individual patient level but also the whole patient-population level (i.e., they capture the heterogeneity observed in patient populations).

Since compounds can be screened simultaneously on a group of patient-relevant models, this enables quicker decision-making when it comes to choosing which lead compound to progress. The matrix HTS approach, which effectively is a clinical trial in a dish, represents a revolutionary approach to early drug discovery. Such high-throughput approaches can now be paired with high-content imaging (HCI) and high-content analysis (HCA) technologies to develop comprehensive cellular profiles of how your compounds are affecting cellular systems and spatial biology.

Combinatorial Drug Strategies in 3D

The identification and translation of effective drug combinations remains challenging, especially due to the difficulties associated with evaluating and quantifying drug combination effects. Combination screens can now be used to evaluate multi-drug strategies across multiple types of 3D in vitro models. For additional information on study design options for drug combination studies, read our previous blog post. In short, while the matrix design is the most complex, it yields highly comprehensive data, including information about drug interactions and dosing across a wider range of ratios as compared to the simpler designs. Figure 1 shows a combination plate map where multiple doses are combined in a 6x6 matrix to assess interactions at multiple dose ratios.


Figure 1: Combination platemap (6x6 matrix).

Two-drug combination effects (synergistic, antagonistic, and additive) can be evaluated and quantified using bioinformatics, such as CrownSyn™ (Crown Bioscience’s bioinformatics service for HTS drug combination analysis). This type of analysis can determine optimal in vivo dosing combinations before proceeding to downstream steps. Figure 2 shows a heatmap and surface plot of a Bliss independence model in synergism analysis which assesses the effects of individual drugs in a combination as independent yet competing events. This information can determine whether the probability of cell death from drug A is statistically independent of the probability of cell death from drug B in a population of cells.


Figure 2: CrownSyn - Heatmap and surface plot of Bliss independence model in synergism analysis.

Matrix designs can be used to evaluate multiple combination strategies across multiple 3D in vitro models, such as tumor organoids. Figure 3 shows the results of combining MK 1775 + MK 8776 to treat the pancreatic cancer (PA5389B) organoid model analyzed by two different methods (Bliss and Loewe). The data show a strong synergy at low concentrations where the synergy score is represented as a 2D contour map, a 3D response surface plot, score, and dose inhibition heat map.


Figure 3: Results of combining MK 1775 + MK 8776 analyzed by two different methods (Bliss and Loewe).

In vitro Multi-drug Combination Screening Assays with Patient-Derived Cystic Fibrosis (CF) Organoids

In this section we describe the results of using Crown Bioscience’s Forskolin Induced Swelling assay to test the effects of the recently approved triple combination therapy TRIKAFTA® (consisting of the correctors VX445 (elexacaftor) and VX661 (tezacaftor) plus the potentiator VX770 (ivacaftor)) on patient-derived Cystic Fibrosis (CF) organoids (denoted as F508del/del).

CF F508del/del organoids were seeded in 384-well plates in 3D and exposed to single CFTR modulators or combinations. Forskolin was added to induce a swelling response. Organoids were quantified with 3D proprietary image analysis software. Figure 4A shows individual dose response curves of the different components of TRIKAFTA. All compounds showed an increase in lumen area, with VX445 (orange) giving the strongest effect. A single dose was then chosen and tested in combination, and these data are shown in Figure 4B. Different effect sizes were observed after addition of the different types of CFTR modulators, with the highest increase observed for the complete TRIKAFTA mixture (final teal bar).


Figure 4: Quantified lumen area after treatment with CFTR modulators as compared to solvent control (0.2% DMSO) and stimulant only control (forskolin). Data presented as the ratio between lumen and organoid areas (avg., N=4).

Representative images of solvent control (untreated, left) and TRIKAFTA treated (right) are shown in Figure 5. Both conditions were stimulated for three hours with forskolin, fixed, stained and analyzed. Fluorescent images (lower left) show nuclei in blue and F-actin in red. Mask images (upper right) represent the outline of the quantified organoids (green) and lumen (red). When untreated, organoids had little to no quantifiable lumen, but swelled after stimulation with forskolin. As shown, after treatment with TRIKAFTA, organoids swelled and presented with a clear lumen.


Figure 5: 3D image analysis in 3D cultured CF F508del/del organoids.

HCI and HCA were used to provide robust quantification of phenotypic changes, allowing accurate response matrix measurements, such as the combination of VX445, VX661 and VX770 in an 8x6x4 matrix in 3D cultured CF F508del/del organoids (Figure 6).


Figure 6: 3D image analysis provides robust quantification of phenotypic changes.
Data are presented as the ratio between lumen and organoid areas (avg., N=4).

3D In Vitro Models in Methylcellulose and Soft Agar Assays using Cell Lines

A range of commercially available in vitro cell lines have been validated for 3D assays, also available at Crown Bioscience. In short, cells are grown, harvested, and seeded on methylcellulose, soft agar, or other commercially available matrices at the appropriate dilution to achieve a final density which has been optimized for each cell line. Figure 7 shows a dose response curve for the head and neck cancer cell line FaDu, grown in 3D and treated with increasing doses of an experimental anticancer compound. Beginning at Day 2, the anticancer compound was administered to each well according to the concentration design and cell growth was monitored daily until the endpoint, when cell numbers were counted using the CellTiter-Glo® (CTG) assay.


Figure 7: Dose Response Curve for the FaDu Head and Neck Cancer Cell Line.

Soft agar assays in low-attachment 96 well plates can also be used. For instance, cell lines can be plated at 3×10³ cells/well in 0.4% agar, over a base layer of 0.6% agar. The test agent can then be added with growth media, and endpoints can be CTG or imaging assays. Figure 8 shows images comparing cisplatin dose response for both of these endpoints. The calculated EC50 values are comparable, which validated HCI as an endpoint for this assay.


Figure 8: Dose Response Curve for CFPAC-1 Cancer Cell Line Treated with Cisplatin Using CTG and HCI.

Ex Vivo Models in 3D Tumor Growth Assays (TGA) using HuPrime® PDX models and PrimePanel™ Cells

By employing highly characterized PDX models, such as Crown Bioscience’s HuPrime PDX models, researchers can reap the benefits of using a clinically relevant in vivo drug efficacy model in combination with 3D in vitro /ex vivo methylcellulose assays that are amenable to high throughput in vitro drug screening (see Figure 9 for the assay principle).


Figure 9: Depiction of how freshly isolated cells from highly-validated PDX models can be used for ex vivo clonogenic assays.

All PDX-derived ex vivo models can be utilized in this assay. For example, for freshly isolated primary cells inoculated in immunodeficient mice, xenografts are harvested, the tumor is lysed, and cells are seeded on methylcellulose or 3D Alvetex® plates to allow growth in 3D. Anticancer agents can then be tested and endpoints for evaluation can include phase contrast imaging and CTG to assess efficacy.

Below is an example of an ex vivo efficacy study conducted on freshly isolated primary cells from the gallbladder tumor model GL0440. Cisplatin was administered at different concentrations and images were taken for each dose.


Figure 10: Ex Vivo Efficacy Study of Cisplatin on Primary Cells Derived from GL0440 Grown in 3D.

In another example, Figure 11 shows the dose response curve for cisplatin using freshly isolated cells from the OV5397 ovarian cancer PDX model in the methylcellulose assay.


Figure 11: Dose Response Curve for Cisplatin Effect on Primary Cells Derived from OV5397 Grown in 3D.

Moving Oncology Models Closer to the Clinic with the Ex Vivo Patient Tissue (EVPT) Platform

The Ex Vivo Patient Tissue (EVPT) platform allows researchers to evaluate anticancer drugs in patient tumors in the context of a preserved native tumor microenvironment (TME), including endogenous immune cells, fibroblasts, and other stromal components. By using 50-300 patient tumor tissues directly seeded in a 384-well format, the EVPT platform is a highly patient-relevant translational system that better recapitulates the heterogeneity and molecular/genetic complexity of human tumors. This allows for better decision-making when it comes to deciding whether to progress your oncology or immuno-oncology therapeutic candidates.

Drug effects, including tumor killing and immune cell proliferation, can be measured using automated 3D phenotypic HCI and HCA for robust evaluation of single and combination treatments in high throughput. Figure 12 depicts the assay principle. Additional analyses can also be conducted using flow cytometry, IHC, cytokine analysis, and next generation sequencing.


Figure 12: EVPT Assay Principle.

The following plot shows the concentration-dependent tumor killing response to various chemotherapeutic drugs using the EVPT platform (ovarian tumor tissue).


Figure 13: Testing Oncology Therapeutics at Various Doses using the EVPT Platform (Ovarian Tumor Tissue).

Figure 14 shows the results of a phenotypic readout for total tumor organoid area as a function of treatment with different immunotherapies regimens (single drugs and combinations).


Figure 14: Immunotherapy Responses with Phenotypic Readouts.

Conclusion

3D in vitro models, such as those described in this post, offer highly patient relevant models that are amenable to high-throughput drug screening for identifying effective dosing strategies. Because these 3D models more closely recapitulate the complex in vivo environment, they allow for better decision-making when it comes to developing dosing strategies, including single or multi-drug combinations. By pairing 3D in vitro studies with HCI and HCA, researchers can develop deep insights into a compound’s effects in vitro while closely mimicking an in vivo environment, ultimately allowing for more accurate predictions of in vivo drug responses.


Related Posts