Mellandagsinlägg - HoloMonitor vid läkemedelsforskning

Halloj alla phi,are. Hoppas ni haft en bra jul med allt det innebär.Snart glider vi in i ett nytt år så undertecknad börjar så smått värma upp motorerna. Här kommer en studie daterad 23/12 2024 som tar upp tekniken vid framtagning av nya läkemedel. Ett gäng forskare från Italien,Danmark och Belgien har studerat hur förbättra forskning och utveckling av nya läkemedel.Man har fokuserat på 2 beståndsdelar som ofta förekommer i cancerläkemedel,nocodazole och paclitaxel.Hur snabbare och effektivare få fram kommande läkemedel. Genom studiens utförande har de haft "kontrollstationer" för att se läkemedlens effektivitet in action på människoceller. PHI`s excellenta HoloMonitor har använts för det syftet.

Forskarna utgår från det faktumet att utveckling av nya läkemedel kostar multum (miljardbelopp) och inte sällan desssvärre inte når hela vägen fram.Ett misslyckande kan alltså slå hårt på även de allra största bjässarna i branschen. De av er som varit med på svenska biotechföretag som sysslar med forskning och gått på pumpen vet konsekvenserna. Därför är behovet av kontrollstationer tidigt in i processen något som kommer gå upp för fler och fler biotechföretag. Men till studien: 

HYDRA: HYdrogel Dispensing with Robotic Automation for high throughput drug testing Posted December 23,2024

Abstract

The poor predictivity of traditional cell culture platforms hampers drug research and development efficiency................

1 Introduction

Predicting how human cells will respond to new therapeutics accurately in preclinical stages is essential for enhancing drug research and development (R&D). However, this process has become notably lengthy, resource-intensive, and risky: typical efforts last ten years, cost over 1 billion US$, and fail nine out of ten times. Genomics and artificial intelligence advancements have led to improvements,but R&D still faces declining productivity and high failure rates in clinical trials.It has been estimated that up to 50% of potential therapeutics that succeed in the initial preclinical assays fail in the following clinical trials, suggesting that existing preclinical models have a low capacity to predict human responses.For example, drug development has relied on cell culture assay using plastic or glass substrates for decades. However, these stiff and inert substrates lack many physicochemical characteristics of soft and active biological tissues. These differences lead to loss of tissue-specific architecture, altered mechanical and metabolic signals, and distinct interactions between cells and their extracellular matrix (ECM). Together, these differences make traditional cell culture platforms poor preclinical models.

Studiens HoloMonitoravsnitt

Drug screening using digital holographic long-term imaging

Nocodazole (M1404-50MG, Merck) and paclitaxel (10461, Cayman Chemical) were chosen as test compounds. The drugs were dissolved in dimethylsulphoxide (D2650-100ML, Merck), aliquoted in 1 mL Eppendorf vial, and kept at −20 °C. 10% w/v FG-based culture platform was fabricated as described previously (see “Preparation of transglutaminase-crosslinked fish gelatin hydrogels” and “Automated hydrogel fabrication using liquid handling robots”). HaCaT cells (P50-P70, 50.000 cells/mL) were seeded on 10% w/v FG hydrogels 24 h before treatment by casting 100 μL per well of the 96-well plate. In the drug screening, cells were treated with three different concentrations per drug, and positive and negative controls (resulting in 12 replicates per group) were included. Specifically, nocodazole and paclitaxel (5 mg/mL) were diluted to 5 μg mL−1 solutions in a DMEM culture medium. Cells were treated with nocodazole concentrations of 12.5, 25, and 50 ng mL−1 and paclitaxel concentrations of 0.5, 2.5, and 12.5 ng mL−1. Wells with 0.1% DMSO and 1500 µg mL−1 Geneticin (10131-027, Gibco) were used as a negative and positive control, respectively. After adding drugs, the 96-well plate was immediately placed on the motorized stage of a HoloMonitor M4 (Phase Holographic Imaging AB) inside a cell incubator (37 °C, 100% humidity, 5% CO2). Using HoloMonitor Appsuite software (Version 4.0.1, Phase Holographic Imaging PHI AB), one field of view per well was selected and imaged every hour for 48 hours using an Olympus PLN 20X objective (NA 0.4) (N1215900, Evident) and a low-power laser unit (635 nm, 0.2 mW cm−2). Time-lapse images were exported and processed on ImageJ FIJI software to extract the cell % confluency. In detail, the cell confluency was defined as the sum of areas occupied by cells / the total size of the field of view) (Figure 4, S6, S7, S8, and Videos s5, s6).

Cell confluency analysis using digital holographic long-term imaging

To extract the cell confluency value (defined as the percentage of the field of view featuring cells), time-lapse datasets from the HoloMonitor microscope were exported from HoloMonitor Appsuite software (Version 4.0.1, Phase Holographic Imaging PHI AB) and processed using the open-source ImageJ FIJI software.

Drug test using phase holographic imaging.

A) Holographic image of HaCaT cultured in 0.1% DMSO – vehicle control – after seeding and B) 48 hours after seeding. C) Holographic image of HaCaT treated with 50 ng mL−1 of nocodazole drug 48 hours after seeding. Scale bars: 50 µm. D) HaCaT cell confluency vs. time. Cells were cultured in nocodazole-based cell culture media (12.5 ng/mL – blue dots, 25 ng/mL – red squares, 50 ng/mL black triangles) for 48 hours on hydrogel substrates. Data were normalized concerning the initial confluency value. Solid lines represent mean values, and shaded areas represent s.e.m (n=12). E) Final cell confluency vs. nocodazole concentration (12.5 ng/mL, 25 ng/mL, 50 ng/mL) on plastic and hydrogel substrates. Data were normalized concerning the initial confluency value. (*) stands for significative difference. F) Nocodazole dose-response curves on plastic (IC50, 16.6 ng mL−1) and hydrogel (IC50, 18.1 ng mL−1) substrates (n=12). Data were normalized concerning the initial confluency value (n=12). Image brightness and contrast were adjusted for printed visibility.

F) Time-lapse holographic images of HaCaT cells on hydrogel substrates acquired during 48 hours of treatment with nocodazole or paclitaxel. Scale bar: 50 μm.

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