Dynamic microphysiological system chip platform for high-throughput,customizable,and multi-dimensional drug screening

Spheroids and organoids have attracted significant attention as innovative models for disease modeling and drug screening.By employing diverse types of spheroids or organoids,it is feasible to establish microphysiological systems that enhance the precision of disease modeling and offer more dependable and comprehensive drug screening.High-throughput microphysiological systems that support optional,parallel testing of multiple drugs have promising applications in personalized medical treatment and drug research.However,establishing such a system is highly challenging and requires a multidisciplinary approach.This study introduces a dynamic Microphysiological System Chip Platform(MSCP)with multiple functional microstructures that encompass the mentioned advantages.We developed a high-throughput lung cancer spheroids model and an intestine-liverheart-lung cancer microphysiological system for conducting parallel testing on four anti-lung cancer drugs,demonstrating the feasibility of the MSCP.This microphysiological system combines microscale and macroscale biomimetics to enable a comprehensive assessment of drug efficacy and side effects.Moreover,the microphysiological system enables evaluation of the real pharmacological effect of drug molecules reaching the target lesion after absorption by normal organs through fluid-based physiological communication.The MSCP could serves as a valuable platform for microphysiological system research,making significant contributions to disease modeling,drug development,and personalized medical treatment.

Phosphorylated protein chip combined with artificial intelligence tools for precise drug screening

We have developed a protein array system,named"Phospho-Totum",which reproduces the phosphorylation state of a sample on the array.The protein array contains 1471 proteins from 273 known signaling pathways.According to the activation degrees of tyrosine kinases in the sample,the corresponding groups of substrate proteins on the array are phosphorylated under the same conditions.In addition to measuring the phosphorylation levels of the 1471 substrates,we have developed and performed the artificial intelligence-assisted tools to further characterize the phosphorylation state and estimate pathway activation,tyrosine kinase activation,and a list of kinase inhibitors that produce phosphorylation states similar to that of the sample.The Phospho-Totum system,which seamlessly links and interrogates the measurements and analyses,has the potential to not only elucidate pathophysiological mechanisms in diseases by reproducing the phosphorylation state of samples,but also be useful for drug discovery,particularly for screening targeted kinases for potential drug kinase inhibitors.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Drug Screening Experiment in vitro of Fox Eperythrozoon

[Objective] The research aimed to make the drug screening experiment in vitro of eperythrozoon of fox. [Method] RPMI-1640 was used as the basic culture medium and 30% calf serum was added. Using Berenil, oxytetracycline, al icin, doxy-cycline,imidocarb,florfenicol,Fuhongjuesha,primaquine phosphate and other drug powder,the drug screening experiment in vitro of fox eperythrozoon was made under the conditions of 37.3 ℃, 5% CO2. [Result] The effects of Fuhongjuesha was the best,and that of primaquine phosphate and Berenil was the next. And imidocarb,al-licin and florfenicol were effective. [Conclusion] The research provided scientific and theoretical basis for the clinical treatment of eperythrozoonosis.

Bioprinting of novel 3D tumor array chip for drug screening

Biomedical field has been seeking a feasible standard drug screening system consisting of 3D tumor model array for drug researching due to providing sufficient samples and simulating actual in vivo tumor growth situation,which is still a challenge to rapidly and uniformly establish though.Here,we propose a novel drug screening system,namely 3D tumor array chip with“layer cake”structure,for drug screening.Accurate gelatin methacryloyl hydrogel droplets(~0.1μL)containing tumor cells can be automatically deposited on demand with electrohydrodynamic 3D printing.Transparent conductive membrane is introduced as a chip basement for preventing charges accumulation during fabricating and convenient observing during screening.Culturing chambers formed by stainless steel and silicon interlayer is convenient to be assembled and recycled.As this chip is compatible with the existing 96-well culturing plate,the drug screening protocols could keep the same as convention.Important properties of this chip,namely printing stability,customizability,accuracy,microenvironment,tumor functionalization,are detailly examined.As a demonstration,it is applied for screening of epirubicin and paclitaxel with breast tumor cells to confirm the compatibility of the proposed screening system with the traditional screening methods.We believe this chip will potentially play a significant role in drug evaluation in the future.

Current Status of Targets and Assays for Anti-HIV Drug Screening

HIV/AIDS is one of the most serious public health challenges globally. Despite the great efforts that are being devoted to prevent,treat and to better understand the disease,it is one of the main causes of morbidity and mortality worldwide. Currently,there are 30 drugs or combinations of drugs approved by FDA. Because of the side-effects,price and drug resistance,it is essential to discover new targets,to develop new technology and to find new anti-HIV drugs. This review summarizes the major targets and assays currently used in anti-HIV drug screening.

A high‑throughput three‑dimensional cell culture platform for drug screening

Traditional two-dimensional(2D)cell cultures lack the extracellular matrix(ECM)-like structure or dynamic fluidic microenvironment for cells to maintain in vivo functionality.Three-dimensional(3D)tissue scaffolds,on the other hand,could provide the ECM-like microenvironment for cells to reformulate into tissue or organoids that are highly useful for in vitro drug screening.In this study,a high-throughput two-chamber 3D microscale tissue model platform is developed.Porous scaffolds are selectively foamed on a commercially available compact disk using laser.Perfusion of cell culture medium is achieved with centrifugal force-driven diffusion by disk rotation.Experimental studies were conducted on the fabrication process under various gas saturation and laser power conditions.Cell cultures were performed with two types of human cell lines:M059K and C3A-sub28.It is shown that the structure of microscale porous scaffolds can be controlled with laser foaming parameters and that coating with polydopamine these scaffolds are inducive for cell attachment and aggregation,forming a 3D network.With many such two-chamber models fabricated on a single CD and perfusion driven by the centrifugal force from rotation,the proposed platform provides a simple solution to the high-cost and lengthy drug development process with a high-throughput and physiologically more relevant tissue model system.

Methodology of drug screening and target identification for new necroptosis inhibitors

Apoptosis has been considered as the only form of regulated cell death for a long time. However, a novel form of programmed cell death called necroptosis was recently reported. The process of necroptosis is regulated and plays a critical role in the occurrence and development of multiple human diseases. Thus,the study on the molecular mechanism of necroptosis and its effective inhibitors has been an attractive field for researchers. Herein, we introduce the molecular mechanism of necroptosis and focus on the literature about necroptosis drug screening in recent years. In addition, the identification of the critical drug targets of the necroptosis is also discussed.

Roles of Community Pharmacists in Screening and Disseminating of Information about Non-Steroidal Anti-Inflammatory Drugs Risks: Implications for Drug Safety Assessment

Background: The increasing use of non-steroidal anti-inflammatory drugs (NSAIDs) both on prescription and over the counter raises a major global health concern because of the risks associated with their use if no proper guidance is given by the health care provider. This study assessed the roles of community pharmacists in screening and disseminating information about the risks associated with NSAID use in Zambia. Methodology: This was a national cross-sectional study in which a structured self-administered questionnaire was administered to 245 registered community pharmacists in Zambia. Stata/BE, version 15.1 (Stata Corporation, College Station, Texas, USA) and multivariate logistic regression model was used to determine factors associated with information dissemination about ADRs of NS-NSAIDs. Results: 231 of the 245 distributed questionnaires were returned giving a response rate of 94.3%. All (100%) participating community pharmacists claimed to have practiced dispensing NSAIDs. However, only 26 (11.0%) and 71 (30.8%) regularly screened for risk factor of selective COX-2 NSAIDS (SC2-NSAIDS) and non-selective NSAIDS (NS-NSAIDs) respectively. Information dissemination on adverse drug reactions (ADRs) of SC2-NSAIDS was regularly provided by only 22 (9.5%) of pharmacists while that of NS-NSAIDs was regularly provided by 49 (21.2%). In the multivariate logistic regression model, being the owner of a pharmacy (AOR: 5.4, CI: 1.84 - 16.4) was significantly associated with information dissemination about ADRs of NS-NSAIDs while an hour increase in the working hours per day (AOR: 0.9, CI: 0.64 - 0.95) was associated with less likelihood of information dissemination. Conclusion: Pharmacists working in community pharmacies in Zambia did not regularly screen and disseminate information about the risks associated with NSAID use. Therefore, pharmacists should be able to screen and monitor patients at risk and be aware of the majority of risk factors while dispensing NSAIDs to minimize the associated complications.

A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50%of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine lesions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glioblastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell characteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening

Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Polyurethane scaffold-based 3D lung cancer model recapitulates in vivo tumor biological behavior for nanoparticulate drug screening

Lung cancer is the leading cause of cancer mortality worldwide.Preclinical studies in lung cancer hold the promise of screening for effective antitumor agents,but mechanistic studies and drug discovery based on 2D cell models have a high failure rate in getting to the clinic.Thus,there is an urgent need to explore more reliable and effective in vitro lung cancer models.Here,we prepared a series of three-dimensional(3D)waterborne biodegradable polyurethane(WBPU)scaffolds as substrates to establish biomimetic tumor models in vitro.These 3D WBPU scaffolds were porous and could absorb large amounts of free water,facilitating the exchange of substances(nutrients and metabolic waste)and cell growth.The scaffolds at wet state could simulate the mechanics(elastic modulus∼1.9 kPa)and morphology(porous structures)of lung tissue and exhibit good biocompatibility.A549 lung cancer cells showed adherent growth pattern and rapidly formed 3D spheroids on WBPU scaffolds.Our results showed that the scaffold-based 3D lung cancer model promoted the expression of anti-apoptotic and epithelial-mesenchymal transition-related genes,giving it a more moderate growth and adhesion pattern compared to 2D cells.In addition,WBPU scaffold-established 3D lung cancer model revealed a closer expression of proteins to in vivo tumor,including tumor stem cell markers,cell proliferation,apoptosis,invasion and tumor resistance proteins.Based on these features,we further demonstrated that the 3D lung cancer model established by the WBPU scaffold was very similar to the in vivo tumor in terms of both resistance and tolerance to nanoparticulate drugs.Taken together,WBPU scaffold-based lung cancer model could better mimic the growth,microenvironment and drug response of tumor in vivo.This emerging 3D culture system holds promise to shorten the formulation cycle of individualized treatments and reduce the use of animals while providing valid research data for clinical trials.

High-Throughput Drug Screening on Borrelia garinii and Borrelia afzelii Identified Hypocrellin A as an Active Drug Candidate Against Borrelia Species

Lyme disease(LD)is a tick-transmitted infection caused by Borrelia burgdorferi sensu lato species,which include B.burgdorferi,Borrelia afzelii and Borrelia garinii.The majority of patients with early LD can be cured by the standard treatment,yet some still suffer from posttreatment LD syndrome.The presence of Borrelia persisters has been proposed as a contributing factor,because they cannot be completely eradicated by the currently used antibiotics for LD.Finding new pharmaceuticals targeting Borrelia persisters is crucial for developing more effective treatments.Here,we first confirmed the existence of persisters in B.garinii and B.afzelii cultures and then conducted a high-throughput screening of a customdrug library against persister-rich stationary-phase B.garinii and B.afzelii cultures.Among 2427 compounds screened,hypocrellin A(HA),anthracycline class of drugs and topical antibiotics along with some other natural compounds were identified to have strong potential for killing persisters of B.garinii and B.afzelii.HA was the most active anti-Borrelia compound,capable of eradicating stationary-phase Borrelia persisters,in particular when combined with doxycycline and/or ceftriaxone.Liposoluble antioxidant vitamin E was found to antagonize the activity of HA,indicating HA’s target is the cell membrane where HA triggers the generation of reactive oxygen species in the presence of light.HA was found to have distinct bactericidal activity against Borrelia species but had poor or no activity against gram-positive and gram-negative bacteria.Identification of the abovementioned drug candidates may help develop more effective therapies for LD.

CRISPR-edited hepatic organoids as drug screening platform for non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD),is a chronic liver disease caused by a build-up of intrahepatic fat.Metabolic comorbidities associated with NAFLD included obesity,type 2 diabetes,hyperlipidemia,hypertension,and metabolic syndrome(1-3).With changes in diet and lifestyle,the incidence of NAFLD is rapidly increasing to an estimated 30%of global population(4).Histologic manifestations of NAFLD range from the mild stage of simple steatosis,to the advanced stage of steatosis accompanied with necroinflammation or fibrosis,so called nonalcoholic non-alcoholic steatohepatitis(NASH).

3D bioprinting of in vitro porous hepatoma models:establishment,evaluation,and anticancer drug testing

Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs.To address this,this study describes the use of threedimensional(3D)bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells(3DP-7721)by combining gelatin methacrylate(GelMA)and poly(ethylene oxide)(PEO)as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells.The GelMA(10%,mass fraction)and PEO(1.6%,mass fraction)hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics,satisfactorymechanical properties,and biocompatibility for the creation of the 3DP-7721 model.Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction(PCR)were used to evaluate the biological properties of the model.Compared with the two-dimensional culture cell model(2D-7721)and the 3D mixed culture cell model(3DM-7721),3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes.Moreover,we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models.In addition,the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice.Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors,the generated solid tumors were similar to actual tumors,and the deterioration was higher.This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.

An Experimental Model for Screening Anti-AIDS Drugs with Bovine Immunodeficiency Virus

The assays for bovine immunodeficiency virus (BIV) induced syncytium formation and BIV long terminal repeat (LTR) directed luciferase (Luc) gene expression were applied to screen and evaluate anti AIDS drugs. Frequency of the syncytium formation and BIV LTR directed Luc activity were in proportion to the number of input BIV infected cells. AZT inhibited the syncytium formation and the BIV LTR directed Luc gene expression level. Its inhibitory effects were dosedependent with the IC 50 being 0.24 and 0.052 mmol / L, respectively.

A new method of immobilizing HEK293 cells on the inner wall of a capillary column as stationary phase for drug screening

It is a new strategy to immobilize cells on the inner wall of a capillary column and use affinity capillary electrophoresis（ACE） to study receptor-ligand interactions or to screen natural products and compounds synthesized by combinatorial chemistry. In this paper, we developed a new method of immobilizing HEK293 cells on the inner wall of a capillary column. Four important experimental conditions were optimized, including cell injection density, PLL concentration, cell culturing time and sterile processing method. Immobilized cell-coated capillary columns prepared under the optimized experimental conditions exhibited good uniformity, stability and durability, which were suitable for capillary electrophoresis. The method could also be used to immobilize HEK293 cells over-expressing certain membrane receptors on the inner wall of a capillary. In this way, cell-coated capillary columns could be applied to ACE drug screening targeting certain membrane proteins.

Fabrication of a hydroxyapatite-PDMS microfluidic chip for bone-related cell culture and drug screening

Bone is an important part of the human body structure and plays a vital role in human health.A microfluidic chip that can simulate the structure and function of bone will provide a platform for bone-related biomedical research.Hydroxyapatite(HA),a bioactive ceramic material,has a similar structure and composition to bone mineralization products.In this study,we used HA as a microfluidic chip component to provide a highly bionic bone environment.HA substrates with different microchannel structures were printed by using ceramic stereolithography(SLA)technology,and the minimum trench width was 50μm.The HA substrate with microchannels was sealed by a thin polydimethylsiloxane(PDMS)layer to make a HA-PDMS microfluidic chip.Cell culture experiments demonstrated that compared with PDMS,HA was more conducive to the proliferation and osteogenic differentiation of the human foetal osteoblast cell line(hFOB).In addition,the concentration gradient of the model drug doxorubicin hydrochloride(DOX)was successfully generated on a Christmas tree structure HA-PDMS chip,and the half maximal inhibitory concentration(IC50)of DOX was determined.The findings of this study indicate that the HA-PDMS microfluidic chip has great potential in the field of high-throughput bonerelated drug screening and bone-related research.

Advances in hydrogel-based vascularized tissues for tissue repair and drug screening

The construction of biomimetic vasculatures within the artificial tissue models or organs is highly required for conveying nutrients,oxygen,and waste products,for improving the survival of engineered tissues in vitro.In recent times,the remarkable progress in utilizing hydrogels and understanding vascular biology have enabled the creation of three-dimensional(3D)tissues and organs composed of highly complex vascular systems.In this review,we give an emphasis on the utilization of hydrogels and their advantages in the vascularization of tissues.Initially,the significance of vascular elements and the regeneration mechanisms of vascularization,including angiogenesis and vasculogenesis,are briefly introduced.Further,we highlight the importance and advantages of hydrogels as artificial microenvironments in fabricating vascularized tissues or organs,in terms of tunable physical properties,high similarity in physiological environments,and alternative shaping mechanisms,among others.Furthermore,we discuss the utilization of such hydrogels-based vascularized tissues in various applications,including tissue regeneration,drug screening,and organ-on-chips.Finally,we put forward the key challenges,including multifunctionalities of hydrogels,selection of suitable cell phenotype,sophisticated engineering techniques,and clinical translation behind the development of the tissues with complex vasculatures towards their future development.

Culture of patient-derived multicellular clusters in suspended hydrogel capsules for pre-clinical personalized drug screening

A personalized medication regimen provides precise treatment for an individual and can be guided by pre-clinical drug screening.The economical and high-efficiency simulation of the liver tumor microenvironment(TME)in a drug-screening model has high value yet challenging to accomplish.Herein,we propose a simulation of the liver TME with suspended alginate-gelatin hydrogel capsules encapsulating patient-derived liver tumor multicellular clusters,and the culture of patient-derived tumor organoids(PDTOs)for personalized pre-clinical drug screening.The hydrogel capsule offers a 3D matrix environment with mechanical and biological properties similar to those of the liver in vivo.As a result,18 of the 28 patient-derived multicellular clusters were successfully cultured as PDTOs.These PDTOs,along with hepatocyte growth factor(HGF)of non-cellular components,preserve stromal cells,including cancer-associated fibroblasts(CAFs)and vascular endothelial cells(VECs).They also maintain stable expression of molecular markers and tumor heterogeneity similar to those of the original liver tumors.Drugs,including cabazitaxel,oxaliplatin,and sorafenib,were tested in PDTOs.The sensitivity of PDTOs to these drugs differs between individuals.The sensitivity of one PDTO to oxaliplatin was validated using magnetic resonance imaging(MRI)and biochemical tests after oxaliplatin clinical treatment of the corresponding patient.Therefore,this approach is promising for economical,accurate,and high-throughput drug screening for personalized treatment.